Study of Cell Proliferation in Hepatocellular Carcinoma and Hepatocyte Transplantation
Date Issued
2005
Date
2005
Author(s)
DOI
zh-TW
Abstract
Hepatocellular carcinoma (HCC) is one of the common malignancies worldwide, and the incidence was relatively higher in south China, Taiwan, southeastern Asia, and sub-Saharan Africa. According to the annual statistics by Department of Health, Executive Yuan, Taiwan, malignant disease is the number one cause of death in 2003 and 2004. Among them, HCC is the number one cause of death in 2003, and the secondary cause of death in 2004. In addition, chronic liver disease including chronic hepatitis, liver cirrhosis that finally resulting in hepatic failure is the number sixth cause of death in 2003, and the number seventh in 2004. Consequently, nearly ten percent of the people in Taiwan died of liver-related disease in the recently two years. At present, liver transplantation is the only available and effective method for treatment of end-staged liver diseases, but the organ donation is still limited. Therefore, researches on both HCC and liver transplantation are imperative, especially in our country.
Part One: Study of Cell Proliferation in Hepatocellular Carcinoma
Section One: Overexpression of REG1A and PAP in Human Hepatocellular Carcinoma
Even though many clinical studies have been made for prognostic prediction in HCC, the overall outcome of patients with HCC has not been completely changed, and specific prognostic indicators are still lacking. HCCs are genetically heterogeneous neoplasm and the genetic heterogeneity correlates with the variety of etiologic factors. Recent studies have shown that many genes that expressed aberrantly in the neoplastic transformation of liver cells had made the HCC differently in tumor invasiveness, metastatic potential, tumor recurrence and patient survival. These factors include p53 gene, angiogenic factors, cell cycle regulators, oncogenes and their receptors, apoptosis related factors, cellular proliferation markers, growth factors, proteinases that involved in the degradation of extracellular matrix, and adhesion molecules.
To identify the candidate genes that expressed differently in human HCC, suppressive subtraction hybridization and microarray techniques were used in this study, and several genes including regenerating islet-derived 1 alpha (REG1A), secreted phosphoprotein 1 (osteopontin), pancreatic lipase-related protein 2 (PNLIPRP2), defender against cell death 1 (DAD1), ATP-binding cassette protein C11 (ABCC11), glycine receptor alpha 1, apolipoprotein B (APOB), dihydropyrimidinase (DPYS), RB1-inducible coiled-coil 1 (RB1CC1), angiotensinogen proteinase inhibitor and so on were discovered. After literatures reviewing, the REG1A was selected to be studied in this study because it’s never been reported in human HCC before. In addition, another gene called pancreatitis-associated protein (PAP) that belongs to the same Reg gene family and located tandemly in the same chromosome region as REG1A was selected in this study because the PAP had been found overexpressed in human HCC but the clinical significance was till unknown. Therefore, in the first portion of this thesis, we studied the expression pattern of these two cell proliferation-related genes in human HCC, and correlated their expression pattern with the clinicopathological features of the patients.
Regenerating gene (Reg), which is expressed prominently in regenerating pancreatic islet, was first identified in the screening of regenerating islet-derived cDNA library taken from 90% depancreatized rat. The Reg gene family consists of a group of antiapoptotic factors or growth factors for pancreatic islet-cells, neural cells and epithelial cells, and was classified into four classes (I~IV) and contained 17 different members till now. In humans, there are two members in Reg I family, REG1A and REG1B. The REGIA gene encodes a 166-amino-acid protein with a 22 amino acids signal sequence, and is highly represented in human pancreatic secretion. The REG1A protein is identical to the pancreatic thread protein, pancreatic stone protein. The REG1B gene codes for a transcript with 87% homology to the REG1A transcript, but the Reg1B protein has never been characterized and its expression in the pancreas remains questionable. Pancreatitis-associated protein (PAP), a member belongs to Reg III family, is also called islet neogenesisassociated protein (INGAP), pancreatic β cell growth factor, na d REG-III. The human PAP cDNA encodes a 175-amino acid protein with 49% identity with the human Reg protein. PAP protein is merely detectable in normal pancreas, but remarkably increased, representing up to 5% of secreted protein in acute pancreatitis and in some chronic pancreatitis, and is also called hepatocarcinoma-intestine-pancreas (HIP) because it can also be detected in intestine and HCC.
In human, the REG1A, REG1B, RS (REG-related sequence), and PAP genes are clustered tandemly in a 95 kb region on chromosome 2p12, and this gene cluster may have arisen from the same ancestral gene by gene duplication. REG I mRNA was expressed in regenerating or hyperplastic pancreatic islets, and expressed high in high-grade biliary dysplasia in hepatolithiasis. REG I was expressed in colorectal cancer, cholangiocarcinoma, and was associated with the infiltrating growth of gastric carcinoma. PAP mRNA was overexpressed during the acute phase of pancreatitis, and can be detected in pituitary adenoma, gastric cancer, colorectal cancer, and was associated with nodal involvement, distant metastasis, and short survival in pancreatic cancer. These indicated that overexpression of REG1A and PAP correlated with invasiveness of some human malignancy. Even though PAP was detected in about a quarter of primary human HCC, the clinicopathological role of REG1A and PAP expression and their interaction in HCC is not clear. In the present study, we demonstrate that PAP expression is associated with a subset of HCCs that is often low-grade, low-stage tumor and shows high frequency of β-catenin mutation, whereas a coexpression of REG1A leads to more advanced disease and poor prognosis.
From January 1983 to December 1997, 1033 surgically resected primary unifocal HCCs were pathologically assessed at the National Taiwan University Hospital. Of these, 265 patients (209 males, 56 females, from 14 to 88 years old with mean age of 55.6 years) who already had mRNA samples taken from resected primary HCC were selected for this study randomly. The tumor grade was divided into three groups, well differentiated (grade I, 61 cases), moderately differentiated (grade II, 102 cases), and poorly differentiated (grade III and IV, 102 cases). HCC tends to spread in the liver via the portal vein invasion even in advanced stage. At the time of operation, no evidence of regional lymph node or distant metastasis was noted, and minute HCC (≦2 cm) has excellent prognosis. HCC with complete fibrous encapsulation has a favorable four-survival, and vascular invasion, the most crucial step of intrahepatic tumor metastasis, is a crucial prognostic factor for HCC. Therefore a modified tumor staging with special emphasis on the extent of vascular invasion, tumor size (≦2 cm or >2 cm), and encapsulation was adopted. Stage I to II HCCs had no vascular invasion, whereas stage III to IV HCCs had various extent of vascular invasion. Stage I HCC (6 case) included completely encapsulated minute HCC ≦2 cm with no liver invasion. Stage II HCC (115 cases) included minute HCC with liver invasion and/or microscopic satellite close to the main tumor; or larger HCC without or with liver invasion and/or minute satellite close to the main tumor. Stage IIIA HCC (43 cases) had invasion of thin-walled vessels in the tumor capsule, but no portal vain invasion or satellite deep in the liver parenchyma. Stage (33 cases) IIIB HCC had invasion of small portal vein in portal tract near the main tumor, but no invasion of major portal vein branch and satellite deep in the liver parenchyma. Stage IV HCC (68 cases) had invasion of major portal vein branches, satellites extending deeply into the surrounding liver, tumor rupture, or invasion of the adjacent organs. The intrahepatic tumor recurrence was based on imaging diagnosis with ultrasonography and/or computed tomography, supplemented with elevated serum α-fetoprotein. Among these patients, 236 (89.1%) were eligible for the evaluation of early intrahepatic tumor recurrence (≦1 year). Twenty-nine patients who died within 1 year after resection and had no information or were negative for intrahepatic tumor recurrence were excluded from the evaluation of early recurrence.
We used RT-PCR for large–scale analysis of PAP and REG1A mRNA levels in 265 unifocal primary HCCs. The primer sequence used was list in Table 1. PAP and REG1A were overexpressed in 97 (36.6%) and 55 (20.8%) tumors, respectively. Both genes were not detectable in 219 nontumorous liver tissues (Fig. 2). PAP overexpression showed a positive correlation with low α-fetoprotein (AFP) level (≦200ng/ml), and low-stage (stage I to II) HCCs that had no vascular invasion, P=0.039 and P=0.013, respectively, whereas REG1A overexpression did not (Table 2). The expression of PAP or REG1A did not correlate with age, gender, chronic hepatitis B infection, chronic hepatitis C infection, tumor size, tumor grade, or early tumor recurrence. In addition, p53 mutation was found in 99 out of 221 cases (44.8%), while β-catenin mutation was detected in 37 out of 248 tumors (14.9%). We found that PAP and REG1A expression showed strong association with β-catenin mutation (P<0.00001 and P=0.00005, respectively), but not with p53 mutation (Table 3).
Because of the frequent coexpression of PAP and REG1A (46 tumors expressed both PAP and REG1A, with a high concordance rate of 77.4%, P<0.00001), we did a combination analysis to further characterize the effects of PAP and REG1A expression in the tumor progression of HCC. We divided these cases into four groups according to presence or absence of PAP and REG1A overexpression: PAP(+)/REG1A(+), PAP(+)/REG1A(-), PAP(-)/REG1A(+), and PAP(-)/REG1A(-). Among the four groups, HCCs expressing PAP alone were associated with the highest frequencies of low-grade (grade I) and low-stage tumors, P<0.007 and P<0.001, respectively, and hence the lowest early tumor recurrence, P=0.051 (Table 4). Consistent with results shown in Table 2, there was also no significant difference in age, gender, α-fetoprotein level, and chronic hepatitis infection between these four groups (data not shown). In the two groups of HCCs with PAP expression, PAP(+)/REG1A(+) HCCs showed more frequent high-grade (84.8% versus 58.8%, P=0.005), high-stage tumors (60.9% versus 29.4%, P=0.002), and hence high early tumor recurrence rate (61.8% versus 31.3%, P=0.006) as compared with PAP(+)/REG1A(-) HCCs (Table 4).
To elucidate the reasons for the more aggressive tumors and early tumor recurrence in PAP(+)/REG1A(+) HCCs than in PAP(+)/REG1A(-) HCCs, we further analyzed the role of p53 and β-catenin mutations. As shown in Table 5, the former group showed significantly higher frequency of p53 mutation (46.2% versus 25.6%, P=0.036), whereas the two groups did not differ significantly inβ-catenin mutation. Then, we further analyzed the role of p53 andβ-catenin mutations in PAP(+)/REG1A(+) and PAP(+)/REG1A(-) HCCs. As shown in Table 6, PAP(+)/REG1A(+) HCCs tended to have more frequent high-stage tumors, but not of statistical significance, and the majority of HCCs withβ-catenin mutation had low-stage tumors, regardless of the presence or absence of REG1A.
Despite the frequent p53 andβ-catenin mutations, approximately half of HCCs are negative for both mutations. We then analyzed potential role of PAP and REG1A expression in this subset of HCC. In the subset of HCCs expressing PAP, the coexpression of REG1A had adverse effect in the groups of HCCs with PAP expression. As shown in Table 6, HCCs with coexpression of REG1A and PAP had more than 3-fold high-stage tumor than those with PAP expression alone (P<0.005). Finally, we analyzed the survival rates of the four different groups of HCCs. According to the expression pattern of PAP and REG1A, HCCs with PAP expression alone had the best 5-year survival, P=0.044, significantly better than HCC with coexpression of PAP and REG1A, P<0.0002 (Fig. 3).
Section Two: Overexpression of LAP18/Stathmin in Human Hepatocellular Carcinoma
In the second portion of the study, we analyzed another gene that also important for proliferation of the cells. Leukemia associated protein 18 (LAP18), also known as stathmin, p18, p19, pp17, prosolin, pp20, 19K, metablastin, and oncoprotein 18, is a ubiquitous cytosolic phosphoprotein, and its expression is regulated during the development in response to extracellular signals regulating cell proliferation, differentiation, and function. LAP18 is proposed to act as a general relay in signal transduction, possibly integrating diverse signals from the cell's environment and also plays an important role in the regulation of microtubule dynamics during the process of cell mitosis. LAP18 was first identified in HL60 leukemic cells, and was overexpressed in acute leukemia, but decreased expression when leukemic cells ceased to proliferate upon exposure to differentiation agents. Expression of LAP18 was most abundant in fetal and adult brain, spinal cord, and cerebellum, followed by thymus, bone marrow, and testis. Expression was intermediate in colon, ovary, placenta, uterus, and trachea, and at substantially lower levels in all other tissues examined. The fetal liver was among the tissue with most abundant LAP18 expression whereas the adult liver had the lowest. In a given tissue, LAP18 was expressed at higher levels in the proliferative compartment than the adjacent more differentiated cells. In addition to the leukemia, higher expression of LAP18 was noted in poorly differentiated solid tumors with high proliferative potential than more differentiated and less proliferative tumors. These observations suggest a strong correlation of LAP18 expression with cellular proliferation in both normal and malignant cells. To identify potential prognostic indicators of human HCC and potential targets for therapeutic strategies, we hypothesized that LAP18 may play a role during liver carcinogenesis. We show here that the LAP18 mRNA is overexpressed in nearly half of human HCC, and the expression correlates with vascular invasion, particularly when cooperation with p53 mutation in HCC.
One hundred and eighty four patients who had adequate and well-preserved RNA samples taken from resected primary HCCs receiving complete pathological assessment were selected for this study including 145 males and 39 females with a mean age of 55.5 years (range, 14-88 years). The tumor grade was divided into three groups, well differentiated (grade I, 39 cases), moderately differentiated (grade II, 79 cases), and poorly differentiated (grade III and IV, 66 cases), and the tumor staging was classified into I (1 case), II (76 cases), IIIA (30 cases), IIIB (28 cases), and IV (49 cases), as previously described. Among them, 165 (88.7%) were eligible for the evaluation of early intrahepatic tumor recurrence (≦1 year). Nineteen patients who died within 1 year after resection and had no information or were negative for intrahepatic tumor recurrence were excluded from the evaluation of early recurrence.
RT-PCR was used for large-scale analysis of LAP18 mRNA expression in HCC and the primer sequence was list in Table 1. Among the 184 unifocal primary HCCs, LAP18 mRNA was overexpressed in 95 tumor specimens (52%, Fig. 4). As shown in Table 7, LAP18 mRNA was overexpressed tended to occur in HCC with HBsAg in sera, P=0.06, but not correlate with age, gender or α-fetoprotein level. Histopathologically, LAP18 overexpression was found more frequently in HCCs with bigger tumor (> 5cm), less differentiation (grade II to IV), and high-stage (stage IIIA to IV) HCCs that had evidence of vascular invasion, P=0.0053, P=0.027 and P=0.0001, respectively. In addition, HCCs with LAP18 overexpression had more frequent early tumor recurrence (P=0.0022), and worse prognosis, with a lower 7-year survival rate than those without LAP18 overexpression, P=0.001 (Fig. 6).
By immunohistochemistry using specific antibody to detect LAP18 protein expression revealed that HCCs with LAP18 mRNA overexpression showed diffuse and intense predominantly cytoplasmic expression of LAP18 protein in the tumor cells. The LAP18-positive tumor cells were often more intensely immunostained at the outer layers of trabeculae, the tumor borders, and intravascular tumor thrombi (Fig. 5A-C). In contrast, LAP18 protein was absent or positive in scattered tumor cells in HCCs without the LAP18 mRNA overexpression (Fig. 5D). The adjacent nontumor livers also showed scattered immunoreactive liver cells with obvious less intense staining.
p53 mutation is the most commonly genetic alternation in HCC, and is known to correlate with tumor aggressiveness and unfavorable prognosis. In this series, p53 mutation was found in 73 out of 158 cases (46%), and p53 mutation showed a positive correlation with high-stage (stage IIIA to IV) HCCs than HCC without p53 mutation, 71% (52/73) versus 42% (36/85), P=0.00028. As shown in Table 7, however, LAP18 overexpression in HCC did not correlate with p53 mutation.
Both LAP18 and p53 were associated with tumor aggressive, and were also important in cell cycle regulation, we then did a combinational analysis to evaluate the potential interplay between these two important unfavorable prognostic factors. We divided these cases into four groups according to presence or absence of Lap18 overexpression and p53 mutation: LAP18(+)/p53(+), 41cases; LAP18(+)/p53(-), 41cases; LAP18(-)/p53(+), 32cases, and LAP18(-)/p53(-), 44cases. As shown in Table 8, LAP18(+)/p53(+) HCCs had the highest frequencies of vascular invasion (stage IIIA to IV) (85%) and early tumor recurrence (68%) than other groups, P=0.00003 and P=0.0035, and hence, the lowest 7-year survival, P<0.016 (Fig. 7). In HCC with p53 mutation, LAP18(+)/p53(+) HCCs had more frequent vascular invasion (stage IIIA~IV) and lower 7-year survival than LAP18(-)/p53(+) HCCs, P=0.0025 and P<0.035, respectively (Table 8; Fig. 7). In HCCs without p53 mutation, however, the frequencies of vascular invasion and prognosis did not differ significantly between HCC with and without LAP18 overexpression.
Part Two: Study of Cell Proliferation in Hepatocyte Transplantation
Section One: Transplantation of Hepatocyte with Augmented Proliferation Capacity
Currently, orthotopic liver transplantation using liver donated by cadaver or living people is the only available method for effective treatment of acquired and genetic-based liver diseases, especially when these diseases reach their end stages. With the limited availability of donor livers, alternative methods have been actively sought through liver cell transplantation. Studies conducted during the 1990s demonstrated that hepatocytes transplanted to the liver, either by injection into the spleen or by infusion through the portal vein, traverse the liver sinusoids and become incorporated into the liver parenchyma. Hepatocyte transplantation is beneficial because of its less invasiveness, easier to be manipulated, viable hepatocytes can be harvested from liver unsuitable for orthotopic liver transplantation, cells from single liver can be used for multiple recipients, and isolated cells can be frozen and distributed shortly when needed. However, the number of cells that can be introduced into the liver by this method is quite limited and engraftment levels are generally below 1.0%. Attempts to increase the proportion of transplanted hepatocytes in the host liver by stimulating liver regeneration are also quite limited because both host and transplanted hepatocytes respond equally to the proliferative stimulus. At present, three experimental models of extensive liver repopulation have been described: the urokinase-type plasminogen activator (uPA) transgenic mouse, the fumarylacetoacetate hydrolase (FAH) null mouse and the retrorsine / partial hepatectomy treated rat. However, in first two models, replacement of liver mass by transplanted hepatocytes is based on continuous and essentially lethal liver injury produced by the genetic disorder, and strong selection of transplanted wild hepatocytes that escape toxic substances damage can proliferate significantly. In the third model, the proliferation of resident hepatocytes were inhibited by toxic substance after partial hepatectomy that offered transplanted hepatocytes time and space for engraftment and proliferation until nearly total liver was replaced. But these animal models were not practical for routine use in human. Therefore, we design a new model to mimic human chronic liver injury with aggravation by acute insult and transplanted hepatocytes with augmented proliferation capacity to evaluate these particular cells in this disease process.
In studies of hepatocyte transplantation, it’s important to distinguish transplanted cells from resident hepatocytes. Many exogenous markers (fluorescent dyes, radioisotopes, et al.) and endogenous markers (transgenes, highly specific molecular probes, reporter genes, retroviral vectors, sex chromosome, et al.) had been used for this purpose, but it’s more convenience using Dipeptidyl peptidase IV (DPPIV) because it can be detected easily by histochemical staining. DPPIV is an ectopeptidase that can specifically cleave X-proline or X-alanine dipeptides from the NH2-terminus of several biological peptides such as the growth hormone-releasing hormone or substance P. Furthermore, DPPIV has been identified as CD26, a cell surface differentiation marker involved in signal transduction in the T-cell lineage. Targeted inactivation of the CD26 gene yielded healthy mice with normal blood level of glucose when fasted and wild hepatocytes containing normal DPPIV activity can be stained red in a membranous bile canalicular distribution pattern. Therefore, it provides as a marker in tracing the transplanted cells in DPPIV knockout mice (Fig. 8).
DPPIV expression was determined on 5-μm-thick cryostat sections from frozen tissue. Fixation was for 5 min in 95% ethanol/5% glacial acetic acid (99:1, v/v) at 0-10°C, followed by a 5-min wash in 95% ethanol at 4°C. Air-dried slides were incubated for 30-40 min at 37°C in the substrate reagent: 100 mg Gly-Pro-4-methoxy-B-naphthylamide (Sigma) dissolved in 6 ml of dimethyl-formamide and mixed with a 100 mg of Fast blue BB salt (Sigma) in 100 ml TMS (0.1 M Tris maleate, 0.1 M NaCI, pH 6.5). The slides were rinsed two times in TMS, incubated for 5 min in 0.1 M CuS04, and rinsed again in TMS. The slides were fixed for 10 min in cold 4% parafonnaldehyde in 0.15 M NaCI, washed two times in 0.15 M NaCI, rinsed in water, counterstained with hematoxylin, and mounted in glycerol.
Multiple sections from both the middle and left liver lobes were taken from each mouse and DPPIV histochemistry staining was performed. Donor hepatocytes were readily identified by DPPIV enzyme expression in a membranous bile canalicular distribution. Ten l00x fields were chosen randomly in sections from each lobe (20 fields were examined for each section) for determination of the number of DPPIV+/+ cell clusters per l00x field and the number of cells present in each cluster. The number of DPPIV4+/+ hepatocytes in each l00x field was then compared to the average total number of hepatocytes per l00x field to determine percent hepatocyte repopulation by transplanted cells. The slides were examined independently by two observers and scored in a blinded fashion by one of the authors.
In the beginning, we have to determine whether the DPPIV knockout mouse can be used as a model to facilitate identification of transplanted DPPIV4+/+ wild hepatocytes, similar to the naturally occurring Fischer 344 DPPIV-/- mutant rat, we transplanted hepatocytes from wild C57B1/6 mice into DPPIV knockout mice in conjunction with two thirds partial hepatectomy, then sacrificed 1,2,3,6 months later to evaluate the transplanted efficiency. As shown in Fig. 11, transplanted hepatocytes, expressing DPPIV in a bile canalicular pattern, engrafted into the periportal regions and became fully integrated into the parenchymal cord structure, and the transplanted cells can remain stable in the host liver for up to 6 months. The small size of DPPIV4 clusters (one, two. or rarely three cells in two-dimensional liver sections) suggested that after their initial engraftment, the transplanted cells underwent one or two divisions during recovery of the animals from partial hepatectomy, but there was no subsequent expansion of transplanted hepatocytes. Thus, the widely used DPPIV transplantation/detection system, originally developed in the rat, can also be used in mice.
In the following study, we attempts to increase the proportion of transplanted hepatocytes by stimulating liver regeneration through repeated CCl4-induced hepatic necrosis after hepatocyte transplantation. Wild type hepatocytes were isolated and transplanted through the spleen into the liver of recipients. The recipients were divided into three groups. Group 1 received wild hepatocyte transplantation only; group 2 received acute CCl4, (1.2 ml/kg mouse body weight) 24 h after wild hepatocyte transplantation; group 3 received acute CCl4 (1.2 ml/kg mouse body weight) 24 h after p27 null hepatocyte transplantation, followed by weekly injections of CCl4 (0.5 ml/kg mouse body weight) for 4 or 8 weeks. Recipients were sacrificed 1 week after the final treatment with CCl4 or vegetable oil only. Liver segments were flash-frozen for histology and DPPIV staining to quantitate donor cell engraftment and proliferation. Even though the repopulation was less than 1%, after 4 weeks, mice receiving either acute insult only (group 2, 0.34 ± 0.10%) or acute insult combined with chronic liver injury (group 3, 0.53 ± 0.23%) had significant liver repopulation than mice absence of a liver regenerative stimulus (group 1, 0.15 ± 0.08%,P = 0.03, and P =0.01, respectively). However, after 8 weeks, the differences became insignificant (group 2, 0.20 ± 0.16% and group 3, 0.46 ± 0.36% versus group 1, 0.05 ± 0.03%; P = 0.09, and P =0.18, respectively). This indicated that liver injury increased the initial engraftment of transplanted hepatocytes but did not augment the proliferation of transplanted hepatocytes.
Then, we transplanted cells capable of higher proliferation activity to evaluate the proliferation of this particular cells in the diseased liver. p27Kipl is a cyclin-dependent kinase inhibitor that stably interacts with cyclin A and cyclin E/Cdk complexes to negatively regulate cell proliferation. p27 null mice are about 25% larger than wild-type littermates and show hyperplasia and organomegaly of multiple organs, including the liver. Hepatocytes in the p27 null liver show accelerated entry into S phase after partial hepatectomy, and p27 inactivation also synergizes with the inactivation of another kinase inhibitor, pi 8, to stimulate hepatocyte proliferation after partial hepatectomy. These results are consistent with previous studies by Karnezis et al., who demonstrated increased [3H]thymidine and BrdU incorporation into DNA in primary hepatocytes from p27 null mice compared with eild hepatocytes.
Because p27 null mice and DPPIV null mice are on different genetic backgrounds, these studies also required development of an immunotolerant DPPIV null mouse capable of accepting allogeneic hepatocytes. This was accomplished by breeding DPPIV null mice with Rag2 null mice, which are deficient in T and B cells due to inability in VDJ rearrangement, and are capable of accepting both allogeneic and xenogeneic cells. DPPIV-/- mice were crossed with Rag2-/- mice to produce heterozygous mice for both DPPIV and Rag2 gene deletions. The double heterozygous mice were mated together to produce either DPPIV-/-Rag2+/- or DPPIV+/- Rag2-/- mice, which were then crossed with each other to generate DPPIV-/- /Rag2-/- double knockout mice. Fig. 12 shows that DPPIV-/- /Rag2-/- mice accepted hepatocytes from various rodent species, including 129sv x C57B1/6 mice, 129sv x CD-1 mice, Balb c mice, and Sprague-Dawley rats, and the transplanted cells can also be identified by DPPIV histochemical staining.
To compare the study that transplanted wild hepatocyte in conjunction with partial hepatectomy, we transplanted P27-/- null hepatocytes into DPPIV-/-/Rag2-/- mice in conjunction with two thirds partial hepatectomy at the time of cell transplantation and followed proliferation of transplanted cells for 6 months (Fig. 13). In contrast to cessation of proliferation by wild hepatocytes in DPPIV-/- mice after 1 month (Fig. 13A), there was a modest increase in the size of DPPIV+/+ cell clusters at 2-3 months after P27+/+ hepatocyte transplantation (Fig. 13B, C), and although infrequent, larger clusters were clearly evident at 6 months after cell transplantation (Fig. 13D).
Thereafter, we attempts to increase the proportion of transplanted hepatocytes by stimulating liver regeneration through repeated CCl4-induced hepatic necrosis after hepatocyte transplantation. For convenience to compare the groups transplanted with p27 null hepatocytes and wild hepatocytes, we divided the mice into three groups and defined as: group 4, received p27 null hepatocyte transplantation only; group 5, received acute CCl4, (1.2 ml/kg mouse body weight) 24 h after p27 null hepatocyte transplantation; and group 6, received acute CCl4 (1.2 ml/kg mouse body weight) 24 h after p27 null hepatocyte transplantation, followed by weekly injections of CCl4 (0.5 ml/kg mouse body weight) for 4 or 8 weeks. Liver segments were also flash-frozen for histology and DPPIV staining to quantitate donor cell engraftment and proliferation.
We found that when an acute liver regenerative stimulus was included at the time of cell transplantation, the P27-/- null hepatocytes repopulated and formed larger clusters than hepatocytes without any regeneration stimulus after 4 weeks (group 5, 0.48 ± 0.23% versus group 4, 0.18 ± 0.17%) and 8 weeks (group 5, 0.40 ± 0.25% versus group 4, 0.10 ± 0.13%), although the differences were small and not statistically significant (P = 0.08, and P =0.15, respectively). However, transplanted cells proliferated and repopulated significantly better in liver with acute liver insult and chronic liver injury than in a normal quiescent liver after 4 weeks (group 6, 1.27 ± 0.76% versus group 4, 0.18 ± 0.17%; P = 0.03), and even better after 8 weeks (group 6, 3.27 ± 2.06% versus group 4, 0.10 ± 0.13%,P = 0.01). In addition, in groups with acute liver insult, P27-/- null hepatocytes repopulated better in those with chronic liver injury after 8 weeks (group 6, 3.27 ± 2.06% versus group 5, 0.40 ± 0.25%,P=0.02) than those with acute insult only, and in mice received acute insult with chronic liver injury, the repopulation of liver increased significantly from 4 weeks to 8 weeks(1.27 ± 0.76%versus 3.27 ± 2.06%, P=0.049).
Further comparison was made between transplantation of p27-/- null and wild hepatocytes. After four weekly doses of CCl4 (Fig. 14), there was no significant increase in the number or size of DPPIV+/+ clusters in livers of mice transplanted with wild hepatocytes (Table 9, Fig. 14A). In contrast, however, after four weekly doses of CCl4, the livers of mice transplanted with p27-/- null hepatocytes showed an increase in both the number and size of DPPIV4 clusters (Table 9. Fig. 14C), and the total number of cells in 100x field increased by two-fold, although the data did not reach statistical significance because of variations between individual animals (22.13 ± 9.00 versus10.27 ± 2.31, P = 0.075). Four additional treatments with CCl4 (a total of eight weekly doses) markedly increased the number and size of DPPIV4 clusters produced by p27 null hepatocytes (Table 9, Fig. 14D), but no such enhancement was observed with p27 wt hepatocytes (Table 9, Fig. 14B). These differences were clearly statistically significant (35.58 ± 16.10 versus 7.53 ± 5.56 in 100x field, P = 0.015).
To quantitate the proliferation of transplanted cells after repeated liver injury, 20 random fields at l00x magnification were examined from multiple sections of each lobe of the liver to determine the average number of clusters per field and the average number of cells per cluster. As showed in Fig. 15, even though the majority of clusters contained one or two cells with both p27 null and wt hepatocytes, the percentage of clusters containing three or more cells was greater with p27 null hepatocytes after four weekly treatments with CCl4 (Fig. 15A). The difference in cluster size between p27 null and wild hepatocytes became much more dramatic after eight weekly treatments with CCl4. Under these conditions, the largest clusters (four in total) derived from wild hepatocytes contained 6-10 cells and none contained more than 10 cells (Fig. 15B). Under comparable circumstances with p27 null hepatocytes, 268 clusters contained 6-10 cells and 110 clusters contained more than 10 cells (Fig. 15B). In addition, the largest clusters derived from p27 null hepatocytes contained 40-50 cells compared with 6-10 cells with p27 wt hepatocytes (cf. Fig. 14D and B). Assuming that each cluster is spherical and derived from a single transplanted cell, the largest clusters with p27 null hepatocytes represent an average of eight cell divisions, whereas those from p27 wt hepatocytes represent an average of five to six cell divisions. These results demonstrate that transplanted p27 null hepatocytes exhibit augmented proliferative activity compared with transplanted wild hepatocytes in the normal host liver and this difference is cumulative during the regenerative response to repeated liver injury.
Liver repopulation by transplanted hepatocytes was measured too. With transplanted hepatocytes alone (quiescent liver), there was no difference in the percent repopulation between p27 null and wt hepatocytes, either at 4 or 8 weeks after cell transplantation. Addition of a single acute liver regenerative stimulus slightly increased liver repopulation by transplanted cells, but again there was no statistically significant difference between p27 null and wild hepatocytes. However, weekly repeated small doses of CCl4 augmented liver repopulation by p27 null hepatocytes versus wild hepatocytes (Fig. 16). After four weekly cycles of CCl4 administration, the extent of liver repopulation by p27 null hepatocytes was more than double that observed with wt hepatocytes, but the difference was still not statistically significant (P=0.14). After eight weekly cycles of CC4 administration, there was a sevenfold greater liver repopulation by p27 null hepatocytes (3.27%, range 1.2-6.1%) compared with wild hepatocytes (0.46%, range 0.04-0.63%) that was highly statistically significant (p= 0.036). In selected areas of high liver repopulation after 8 weeks of CC l4 treatment (predominantly in the periportal and midzonal regions), p27 null hepatocytes replaced up to 12-15% of total hepatic mass compared with 1-1.5% with wild hepatocytes. These results all indicated that p27 null hepatocyte still under normal control in regenerated liver and can not proliferate spontaneously and indefinitely after liver regeneration finished. On the other hand, chronic liver injury enhanced the proliferation capacity of the p27 null hepatocytes, resulted in augmentation of liver repopulation.
Section Two: Chronic Liver Injury before Cell Transplantation Attenuate Function of Hepatic Kupffer cell and Enhanced Liver Repopulation
Last but not least, we want to find out the effect of preceding chronic liver disease on the proliferation capacity of transplanted cells. In the mean time, we developed an animal model using acetaminophen, a very common analgesic, to induce acute liver injury because acetaminophen poisoning is very common in human. In the UK 50% of poisoning admissions involve acetaminophen. In the United State, acetaminophen intoxication accounts for nearer 10% poisoning admissions and that resulted in acute liver failure in as many as 800 people, and one-third of whom died every year. The major pathways of acetaminophen in liver include glucuronidation or sulphation, and the resulting non-toxic conjugates excreted by the kidney. In addition, another pathway involves the cytochrome P-450 (CYP) system, especially CYP2E1, by which acetaminophen is metabolized to the highly reactive metabolite N-acetyl-p- benzoquinoneimine, that may bind covalently with hepatic proteins causing cellular necrosis. The toxic effect of N-acetylp-benzoquinoneimine can be eliminated by the natural antidote glutathione. When taken overdose, acetaminophen will cause a potentially fatal, hepatic centrilobular necrosis. Acute liver failure induced by acetaminophen is a multisystem disorder, with acute renal failure, hypotension, sepsis, coagulopathy, encephalopathy and cerebral edema. Patients who present within 24 h of an acetaminophen overdose can be managed medically. But for those who developed acute liver failure, meticulous supportive care and transplantation is considered for those patients who will die without liver transplantation.
In the fourth portion, we transplanted wild hepatocytes into acetaminophen intoxicated mice without (group 1) or with (group 2) repeated CCl4 injury twice a week for 4 weeks before cell transplantation, and then to sacrifice the mice 7 and 14 days after the transplantation to evaluate the repopulation capacity of transplanted cells.
We found that after chronic repeated liver regenerative stimulus before cell transplantation, number and cluster number of the transplanted cell in 100x field were larger than those without repeated regenerative stimulus at 7 days (21.53 ± 6.29 versus 8.25 ± 3.98, and 14.86 ± 4.62 versus 6.59 ± 3.39; P<0.001 and P<0.001, respectively; Fig. 17, Table 10). The cell number and cluster number remained larger significantly in group with repeated CCl4 injury at 14 days too (17.29 ± 4.40 versus 10.16 ± 3.31, 12.59 ± 3.19 versus 7.90 ± 1.58; P=0.001, and P=0.004, respectively, Table 10), and therefore better hepatocyte repopulation (0.44% versus 0.16% at 7 days, and 0.35% versus 0.20% at 14 days). However, the cell number, the cluster number and repopulation decreased from 7 days to 14 days in liver with repeated CCl4 injury.
Even though the percentage of clusters containing three or more cells was greater in liver with repeated chronic injury at 7 days (P=0.007), the majority of clusters still contained only one or two cells, and the percentage of cluster containing three or more cells were similar 14 days after transplantation. This indicated that repeated chronic liver injury increased engraftment and stimulated initial proliferation of transplanted cells but the proliferation ability is limited after hepatocyte transplantation without further stimulation.
After cell transplantation, the transplanted cell deposited in the hepatic sinusoid, and the Kupffer cells are activated shortly. Therefore, a significant fraction of transplanted cells were destroyed especially when the transplanted cells are entrapped in portal spaces. It’s also reported that perturbation of Kupffer cells activity might increase engraftment of transplanted cell. To elucidate the role of hepatic macrophage on the engraftment of transplanted cell, we use fluorescence immunohistochemical staining to assess the distribution, and use lipopolysaccharide (LPS) stimulation to evaluate the activity of the hepatic macrophage. The hepatic macrophage distributed scatter throughout the plate in quiescent liver. After acute acetaminophen intoxication, the number of macrophage increased but still exhibited generalized distribution. After repeated chronic liver injury, the hepatic macrophages increased and were distributed prominently in the portal area, and even high up after additional acetaminophen insult (Fig. 18). Before LPS stimulation, the TNF-α mRNA producing ability of the Kupffer cells isolated from mice treated with acute on chronic liver injury was prominent than those from acute injury only (TNF-αmRNA/GAPDH level, 117.8 ± 12.3 versus 73.9 ± 12.4, P=0.012). But after LPS stimulation, macrophages isolated from mice that treated with acute on chronic liver injury had the TNF-α mRNA producing ability significantly lower than those from mice receiving acute acetaminophen only (TNF-αmRNA/GAPDH level, 175.7 ± 54.6 versus 465.6 ± 54.2, P=0.004: Fig. 20). It indicated that chronic repeated liver injury before cell transplantation attenuated the function of hepatic Kupffer cells, and might contribute to increased engraftment of transplanted cells. But the molecular relationship between transplanted cell engraftment and Kupffer cell function deserved further evaluation.
In this study, we demonstrated that REG1A, PAP and LAP18 were all correlated with cell proliferation and tumor invasiveness in hepatocellular carcinoma. We showed that both REG1A and PAP were correlated well with β-catenin mutation, but HCCs with PAP expression alone had the highest frequencies of well differentiated and low-stage tumors, the least frequent early tumor recurrence, and the best 5-year survival, while HCC coexpressed with REG1A is associated with more advanced disease and may contribute to tumor progression. In addition, we showed that LAP18 overexpression was associated with bigger tumor, high-grade, high-stage HCC with vascular invasion, more frequent early tumor recurrence, and worse prognosis. These findings suggest that LAP18 confers growth advantage and facilitates tumor cell growth and invasion capacity. These observations provide novel in vivo evidence of PAP, REG1A and LAP18 on cell proliferation capacity in hepatocellular carcinoma, and might offer specific targets for therapeutic strategies concerning cell proliferation in hepatocellular carcinoma.
Furthermore, we demonstrated augmented hepatocyte transplantation with genetic alternated hepatocytes in vivo. Our results revealed that, p27 null hepatocytes did not differ from their wild counterpart in their engraftment or proliferative activity in quiescent livers, and exhibited a slightly proliferative advantage under an acute proliferative stimulus. This suggests that once the liver mass has returned to normal, the p27 null hepatocytes cannot overcome the normal growth control, but the genetic altered hepatocytes can proliferate significantly after repeated liver regeneration stimulus. However, uncontrolled proliferation often impairs cellular differentiation and can pose unacceptable tumor risk. Therefore, a delicate balance should be achieved that would augment cellular proliferation and benefit transplanted hepatocytes, but would not increase tumor risk by the transplanted cells.
Finally, we demonstrate that chronic liver disease increased engraftment of transplanted cells and this phenomenon might result from attenuation of resident Kupffer cells in the recipient liver. This indicated that instead of doing harm for the patient, chronic liver disease might be beneficial for transplanted cells to be engrafted and repopulated in the liver. Additional studies to attenuate the function of hepatic Kupffer cells and increase the repopulation of transplanted cells are mandatory in the near future.
Part One: Study of Cell Proliferation in Hepatocellular Carcinoma
Section One: Overexpression of REG1A and PAP in Human Hepatocellular Carcinoma
Even though many clinical studies have been made for prognostic prediction in HCC, the overall outcome of patients with HCC has not been completely changed, and specific prognostic indicators are still lacking. HCCs are genetically heterogeneous neoplasm and the genetic heterogeneity correlates with the variety of etiologic factors. Recent studies have shown that many genes that expressed aberrantly in the neoplastic transformation of liver cells had made the HCC differently in tumor invasiveness, metastatic potential, tumor recurrence and patient survival. These factors include p53 gene, angiogenic factors, cell cycle regulators, oncogenes and their receptors, apoptosis related factors, cellular proliferation markers, growth factors, proteinases that involved in the degradation of extracellular matrix, and adhesion molecules.
To identify the candidate genes that expressed differently in human HCC, suppressive subtraction hybridization and microarray techniques were used in this study, and several genes including regenerating islet-derived 1 alpha (REG1A), secreted phosphoprotein 1 (osteopontin), pancreatic lipase-related protein 2 (PNLIPRP2), defender against cell death 1 (DAD1), ATP-binding cassette protein C11 (ABCC11), glycine receptor alpha 1, apolipoprotein B (APOB), dihydropyrimidinase (DPYS), RB1-inducible coiled-coil 1 (RB1CC1), angiotensinogen proteinase inhibitor and so on were discovered. After literatures reviewing, the REG1A was selected to be studied in this study because it’s never been reported in human HCC before. In addition, another gene called pancreatitis-associated protein (PAP) that belongs to the same Reg gene family and located tandemly in the same chromosome region as REG1A was selected in this study because the PAP had been found overexpressed in human HCC but the clinical significance was till unknown. Therefore, in the first portion of this thesis, we studied the expression pattern of these two cell proliferation-related genes in human HCC, and correlated their expression pattern with the clinicopathological features of the patients.
Regenerating gene (Reg), which is expressed prominently in regenerating pancreatic islet, was first identified in the screening of regenerating islet-derived cDNA library taken from 90% depancreatized rat. The Reg gene family consists of a group of antiapoptotic factors or growth factors for pancreatic islet-cells, neural cells and epithelial cells, and was classified into four classes (I~IV) and contained 17 different members till now. In humans, there are two members in Reg I family, REG1A and REG1B. The REGIA gene encodes a 166-amino-acid protein with a 22 amino acids signal sequence, and is highly represented in human pancreatic secretion. The REG1A protein is identical to the pancreatic thread protein, pancreatic stone protein. The REG1B gene codes for a transcript with 87% homology to the REG1A transcript, but the Reg1B protein has never been characterized and its expression in the pancreas remains questionable. Pancreatitis-associated protein (PAP), a member belongs to Reg III family, is also called islet neogenesisassociated protein (INGAP), pancreatic β cell growth factor, na d REG-III. The human PAP cDNA encodes a 175-amino acid protein with 49% identity with the human Reg protein. PAP protein is merely detectable in normal pancreas, but remarkably increased, representing up to 5% of secreted protein in acute pancreatitis and in some chronic pancreatitis, and is also called hepatocarcinoma-intestine-pancreas (HIP) because it can also be detected in intestine and HCC.
In human, the REG1A, REG1B, RS (REG-related sequence), and PAP genes are clustered tandemly in a 95 kb region on chromosome 2p12, and this gene cluster may have arisen from the same ancestral gene by gene duplication. REG I mRNA was expressed in regenerating or hyperplastic pancreatic islets, and expressed high in high-grade biliary dysplasia in hepatolithiasis. REG I was expressed in colorectal cancer, cholangiocarcinoma, and was associated with the infiltrating growth of gastric carcinoma. PAP mRNA was overexpressed during the acute phase of pancreatitis, and can be detected in pituitary adenoma, gastric cancer, colorectal cancer, and was associated with nodal involvement, distant metastasis, and short survival in pancreatic cancer. These indicated that overexpression of REG1A and PAP correlated with invasiveness of some human malignancy. Even though PAP was detected in about a quarter of primary human HCC, the clinicopathological role of REG1A and PAP expression and their interaction in HCC is not clear. In the present study, we demonstrate that PAP expression is associated with a subset of HCCs that is often low-grade, low-stage tumor and shows high frequency of β-catenin mutation, whereas a coexpression of REG1A leads to more advanced disease and poor prognosis.
From January 1983 to December 1997, 1033 surgically resected primary unifocal HCCs were pathologically assessed at the National Taiwan University Hospital. Of these, 265 patients (209 males, 56 females, from 14 to 88 years old with mean age of 55.6 years) who already had mRNA samples taken from resected primary HCC were selected for this study randomly. The tumor grade was divided into three groups, well differentiated (grade I, 61 cases), moderately differentiated (grade II, 102 cases), and poorly differentiated (grade III and IV, 102 cases). HCC tends to spread in the liver via the portal vein invasion even in advanced stage. At the time of operation, no evidence of regional lymph node or distant metastasis was noted, and minute HCC (≦2 cm) has excellent prognosis. HCC with complete fibrous encapsulation has a favorable four-survival, and vascular invasion, the most crucial step of intrahepatic tumor metastasis, is a crucial prognostic factor for HCC. Therefore a modified tumor staging with special emphasis on the extent of vascular invasion, tumor size (≦2 cm or >2 cm), and encapsulation was adopted. Stage I to II HCCs had no vascular invasion, whereas stage III to IV HCCs had various extent of vascular invasion. Stage I HCC (6 case) included completely encapsulated minute HCC ≦2 cm with no liver invasion. Stage II HCC (115 cases) included minute HCC with liver invasion and/or microscopic satellite close to the main tumor; or larger HCC without or with liver invasion and/or minute satellite close to the main tumor. Stage IIIA HCC (43 cases) had invasion of thin-walled vessels in the tumor capsule, but no portal vain invasion or satellite deep in the liver parenchyma. Stage (33 cases) IIIB HCC had invasion of small portal vein in portal tract near the main tumor, but no invasion of major portal vein branch and satellite deep in the liver parenchyma. Stage IV HCC (68 cases) had invasion of major portal vein branches, satellites extending deeply into the surrounding liver, tumor rupture, or invasion of the adjacent organs. The intrahepatic tumor recurrence was based on imaging diagnosis with ultrasonography and/or computed tomography, supplemented with elevated serum α-fetoprotein. Among these patients, 236 (89.1%) were eligible for the evaluation of early intrahepatic tumor recurrence (≦1 year). Twenty-nine patients who died within 1 year after resection and had no information or were negative for intrahepatic tumor recurrence were excluded from the evaluation of early recurrence.
We used RT-PCR for large–scale analysis of PAP and REG1A mRNA levels in 265 unifocal primary HCCs. The primer sequence used was list in Table 1. PAP and REG1A were overexpressed in 97 (36.6%) and 55 (20.8%) tumors, respectively. Both genes were not detectable in 219 nontumorous liver tissues (Fig. 2). PAP overexpression showed a positive correlation with low α-fetoprotein (AFP) level (≦200ng/ml), and low-stage (stage I to II) HCCs that had no vascular invasion, P=0.039 and P=0.013, respectively, whereas REG1A overexpression did not (Table 2). The expression of PAP or REG1A did not correlate with age, gender, chronic hepatitis B infection, chronic hepatitis C infection, tumor size, tumor grade, or early tumor recurrence. In addition, p53 mutation was found in 99 out of 221 cases (44.8%), while β-catenin mutation was detected in 37 out of 248 tumors (14.9%). We found that PAP and REG1A expression showed strong association with β-catenin mutation (P<0.00001 and P=0.00005, respectively), but not with p53 mutation (Table 3).
Because of the frequent coexpression of PAP and REG1A (46 tumors expressed both PAP and REG1A, with a high concordance rate of 77.4%, P<0.00001), we did a combination analysis to further characterize the effects of PAP and REG1A expression in the tumor progression of HCC. We divided these cases into four groups according to presence or absence of PAP and REG1A overexpression: PAP(+)/REG1A(+), PAP(+)/REG1A(-), PAP(-)/REG1A(+), and PAP(-)/REG1A(-). Among the four groups, HCCs expressing PAP alone were associated with the highest frequencies of low-grade (grade I) and low-stage tumors, P<0.007 and P<0.001, respectively, and hence the lowest early tumor recurrence, P=0.051 (Table 4). Consistent with results shown in Table 2, there was also no significant difference in age, gender, α-fetoprotein level, and chronic hepatitis infection between these four groups (data not shown). In the two groups of HCCs with PAP expression, PAP(+)/REG1A(+) HCCs showed more frequent high-grade (84.8% versus 58.8%, P=0.005), high-stage tumors (60.9% versus 29.4%, P=0.002), and hence high early tumor recurrence rate (61.8% versus 31.3%, P=0.006) as compared with PAP(+)/REG1A(-) HCCs (Table 4).
To elucidate the reasons for the more aggressive tumors and early tumor recurrence in PAP(+)/REG1A(+) HCCs than in PAP(+)/REG1A(-) HCCs, we further analyzed the role of p53 and β-catenin mutations. As shown in Table 5, the former group showed significantly higher frequency of p53 mutation (46.2% versus 25.6%, P=0.036), whereas the two groups did not differ significantly inβ-catenin mutation. Then, we further analyzed the role of p53 andβ-catenin mutations in PAP(+)/REG1A(+) and PAP(+)/REG1A(-) HCCs. As shown in Table 6, PAP(+)/REG1A(+) HCCs tended to have more frequent high-stage tumors, but not of statistical significance, and the majority of HCCs withβ-catenin mutation had low-stage tumors, regardless of the presence or absence of REG1A.
Despite the frequent p53 andβ-catenin mutations, approximately half of HCCs are negative for both mutations. We then analyzed potential role of PAP and REG1A expression in this subset of HCC. In the subset of HCCs expressing PAP, the coexpression of REG1A had adverse effect in the groups of HCCs with PAP expression. As shown in Table 6, HCCs with coexpression of REG1A and PAP had more than 3-fold high-stage tumor than those with PAP expression alone (P<0.005). Finally, we analyzed the survival rates of the four different groups of HCCs. According to the expression pattern of PAP and REG1A, HCCs with PAP expression alone had the best 5-year survival, P=0.044, significantly better than HCC with coexpression of PAP and REG1A, P<0.0002 (Fig. 3).
Section Two: Overexpression of LAP18/Stathmin in Human Hepatocellular Carcinoma
In the second portion of the study, we analyzed another gene that also important for proliferation of the cells. Leukemia associated protein 18 (LAP18), also known as stathmin, p18, p19, pp17, prosolin, pp20, 19K, metablastin, and oncoprotein 18, is a ubiquitous cytosolic phosphoprotein, and its expression is regulated during the development in response to extracellular signals regulating cell proliferation, differentiation, and function. LAP18 is proposed to act as a general relay in signal transduction, possibly integrating diverse signals from the cell's environment and also plays an important role in the regulation of microtubule dynamics during the process of cell mitosis. LAP18 was first identified in HL60 leukemic cells, and was overexpressed in acute leukemia, but decreased expression when leukemic cells ceased to proliferate upon exposure to differentiation agents. Expression of LAP18 was most abundant in fetal and adult brain, spinal cord, and cerebellum, followed by thymus, bone marrow, and testis. Expression was intermediate in colon, ovary, placenta, uterus, and trachea, and at substantially lower levels in all other tissues examined. The fetal liver was among the tissue with most abundant LAP18 expression whereas the adult liver had the lowest. In a given tissue, LAP18 was expressed at higher levels in the proliferative compartment than the adjacent more differentiated cells. In addition to the leukemia, higher expression of LAP18 was noted in poorly differentiated solid tumors with high proliferative potential than more differentiated and less proliferative tumors. These observations suggest a strong correlation of LAP18 expression with cellular proliferation in both normal and malignant cells. To identify potential prognostic indicators of human HCC and potential targets for therapeutic strategies, we hypothesized that LAP18 may play a role during liver carcinogenesis. We show here that the LAP18 mRNA is overexpressed in nearly half of human HCC, and the expression correlates with vascular invasion, particularly when cooperation with p53 mutation in HCC.
One hundred and eighty four patients who had adequate and well-preserved RNA samples taken from resected primary HCCs receiving complete pathological assessment were selected for this study including 145 males and 39 females with a mean age of 55.5 years (range, 14-88 years). The tumor grade was divided into three groups, well differentiated (grade I, 39 cases), moderately differentiated (grade II, 79 cases), and poorly differentiated (grade III and IV, 66 cases), and the tumor staging was classified into I (1 case), II (76 cases), IIIA (30 cases), IIIB (28 cases), and IV (49 cases), as previously described. Among them, 165 (88.7%) were eligible for the evaluation of early intrahepatic tumor recurrence (≦1 year). Nineteen patients who died within 1 year after resection and had no information or were negative for intrahepatic tumor recurrence were excluded from the evaluation of early recurrence.
RT-PCR was used for large-scale analysis of LAP18 mRNA expression in HCC and the primer sequence was list in Table 1. Among the 184 unifocal primary HCCs, LAP18 mRNA was overexpressed in 95 tumor specimens (52%, Fig. 4). As shown in Table 7, LAP18 mRNA was overexpressed tended to occur in HCC with HBsAg in sera, P=0.06, but not correlate with age, gender or α-fetoprotein level. Histopathologically, LAP18 overexpression was found more frequently in HCCs with bigger tumor (> 5cm), less differentiation (grade II to IV), and high-stage (stage IIIA to IV) HCCs that had evidence of vascular invasion, P=0.0053, P=0.027 and P=0.0001, respectively. In addition, HCCs with LAP18 overexpression had more frequent early tumor recurrence (P=0.0022), and worse prognosis, with a lower 7-year survival rate than those without LAP18 overexpression, P=0.001 (Fig. 6).
By immunohistochemistry using specific antibody to detect LAP18 protein expression revealed that HCCs with LAP18 mRNA overexpression showed diffuse and intense predominantly cytoplasmic expression of LAP18 protein in the tumor cells. The LAP18-positive tumor cells were often more intensely immunostained at the outer layers of trabeculae, the tumor borders, and intravascular tumor thrombi (Fig. 5A-C). In contrast, LAP18 protein was absent or positive in scattered tumor cells in HCCs without the LAP18 mRNA overexpression (Fig. 5D). The adjacent nontumor livers also showed scattered immunoreactive liver cells with obvious less intense staining.
p53 mutation is the most commonly genetic alternation in HCC, and is known to correlate with tumor aggressiveness and unfavorable prognosis. In this series, p53 mutation was found in 73 out of 158 cases (46%), and p53 mutation showed a positive correlation with high-stage (stage IIIA to IV) HCCs than HCC without p53 mutation, 71% (52/73) versus 42% (36/85), P=0.00028. As shown in Table 7, however, LAP18 overexpression in HCC did not correlate with p53 mutation.
Both LAP18 and p53 were associated with tumor aggressive, and were also important in cell cycle regulation, we then did a combinational analysis to evaluate the potential interplay between these two important unfavorable prognostic factors. We divided these cases into four groups according to presence or absence of Lap18 overexpression and p53 mutation: LAP18(+)/p53(+), 41cases; LAP18(+)/p53(-), 41cases; LAP18(-)/p53(+), 32cases, and LAP18(-)/p53(-), 44cases. As shown in Table 8, LAP18(+)/p53(+) HCCs had the highest frequencies of vascular invasion (stage IIIA to IV) (85%) and early tumor recurrence (68%) than other groups, P=0.00003 and P=0.0035, and hence, the lowest 7-year survival, P<0.016 (Fig. 7). In HCC with p53 mutation, LAP18(+)/p53(+) HCCs had more frequent vascular invasion (stage IIIA~IV) and lower 7-year survival than LAP18(-)/p53(+) HCCs, P=0.0025 and P<0.035, respectively (Table 8; Fig. 7). In HCCs without p53 mutation, however, the frequencies of vascular invasion and prognosis did not differ significantly between HCC with and without LAP18 overexpression.
Part Two: Study of Cell Proliferation in Hepatocyte Transplantation
Section One: Transplantation of Hepatocyte with Augmented Proliferation Capacity
Currently, orthotopic liver transplantation using liver donated by cadaver or living people is the only available method for effective treatment of acquired and genetic-based liver diseases, especially when these diseases reach their end stages. With the limited availability of donor livers, alternative methods have been actively sought through liver cell transplantation. Studies conducted during the 1990s demonstrated that hepatocytes transplanted to the liver, either by injection into the spleen or by infusion through the portal vein, traverse the liver sinusoids and become incorporated into the liver parenchyma. Hepatocyte transplantation is beneficial because of its less invasiveness, easier to be manipulated, viable hepatocytes can be harvested from liver unsuitable for orthotopic liver transplantation, cells from single liver can be used for multiple recipients, and isolated cells can be frozen and distributed shortly when needed. However, the number of cells that can be introduced into the liver by this method is quite limited and engraftment levels are generally below 1.0%. Attempts to increase the proportion of transplanted hepatocytes in the host liver by stimulating liver regeneration are also quite limited because both host and transplanted hepatocytes respond equally to the proliferative stimulus. At present, three experimental models of extensive liver repopulation have been described: the urokinase-type plasminogen activator (uPA) transgenic mouse, the fumarylacetoacetate hydrolase (FAH) null mouse and the retrorsine / partial hepatectomy treated rat. However, in first two models, replacement of liver mass by transplanted hepatocytes is based on continuous and essentially lethal liver injury produced by the genetic disorder, and strong selection of transplanted wild hepatocytes that escape toxic substances damage can proliferate significantly. In the third model, the proliferation of resident hepatocytes were inhibited by toxic substance after partial hepatectomy that offered transplanted hepatocytes time and space for engraftment and proliferation until nearly total liver was replaced. But these animal models were not practical for routine use in human. Therefore, we design a new model to mimic human chronic liver injury with aggravation by acute insult and transplanted hepatocytes with augmented proliferation capacity to evaluate these particular cells in this disease process.
In studies of hepatocyte transplantation, it’s important to distinguish transplanted cells from resident hepatocytes. Many exogenous markers (fluorescent dyes, radioisotopes, et al.) and endogenous markers (transgenes, highly specific molecular probes, reporter genes, retroviral vectors, sex chromosome, et al.) had been used for this purpose, but it’s more convenience using Dipeptidyl peptidase IV (DPPIV) because it can be detected easily by histochemical staining. DPPIV is an ectopeptidase that can specifically cleave X-proline or X-alanine dipeptides from the NH2-terminus of several biological peptides such as the growth hormone-releasing hormone or substance P. Furthermore, DPPIV has been identified as CD26, a cell surface differentiation marker involved in signal transduction in the T-cell lineage. Targeted inactivation of the CD26 gene yielded healthy mice with normal blood level of glucose when fasted and wild hepatocytes containing normal DPPIV activity can be stained red in a membranous bile canalicular distribution pattern. Therefore, it provides as a marker in tracing the transplanted cells in DPPIV knockout mice (Fig. 8).
DPPIV expression was determined on 5-μm-thick cryostat sections from frozen tissue. Fixation was for 5 min in 95% ethanol/5% glacial acetic acid (99:1, v/v) at 0-10°C, followed by a 5-min wash in 95% ethanol at 4°C. Air-dried slides were incubated for 30-40 min at 37°C in the substrate reagent: 100 mg Gly-Pro-4-methoxy-B-naphthylamide (Sigma) dissolved in 6 ml of dimethyl-formamide and mixed with a 100 mg of Fast blue BB salt (Sigma) in 100 ml TMS (0.1 M Tris maleate, 0.1 M NaCI, pH 6.5). The slides were rinsed two times in TMS, incubated for 5 min in 0.1 M CuS04, and rinsed again in TMS. The slides were fixed for 10 min in cold 4% parafonnaldehyde in 0.15 M NaCI, washed two times in 0.15 M NaCI, rinsed in water, counterstained with hematoxylin, and mounted in glycerol.
Multiple sections from both the middle and left liver lobes were taken from each mouse and DPPIV histochemistry staining was performed. Donor hepatocytes were readily identified by DPPIV enzyme expression in a membranous bile canalicular distribution. Ten l00x fields were chosen randomly in sections from each lobe (20 fields were examined for each section) for determination of the number of DPPIV+/+ cell clusters per l00x field and the number of cells present in each cluster. The number of DPPIV4+/+ hepatocytes in each l00x field was then compared to the average total number of hepatocytes per l00x field to determine percent hepatocyte repopulation by transplanted cells. The slides were examined independently by two observers and scored in a blinded fashion by one of the authors.
In the beginning, we have to determine whether the DPPIV knockout mouse can be used as a model to facilitate identification of transplanted DPPIV4+/+ wild hepatocytes, similar to the naturally occurring Fischer 344 DPPIV-/- mutant rat, we transplanted hepatocytes from wild C57B1/6 mice into DPPIV knockout mice in conjunction with two thirds partial hepatectomy, then sacrificed 1,2,3,6 months later to evaluate the transplanted efficiency. As shown in Fig. 11, transplanted hepatocytes, expressing DPPIV in a bile canalicular pattern, engrafted into the periportal regions and became fully integrated into the parenchymal cord structure, and the transplanted cells can remain stable in the host liver for up to 6 months. The small size of DPPIV4 clusters (one, two. or rarely three cells in two-dimensional liver sections) suggested that after their initial engraftment, the transplanted cells underwent one or two divisions during recovery of the animals from partial hepatectomy, but there was no subsequent expansion of transplanted hepatocytes. Thus, the widely used DPPIV transplantation/detection system, originally developed in the rat, can also be used in mice.
In the following study, we attempts to increase the proportion of transplanted hepatocytes by stimulating liver regeneration through repeated CCl4-induced hepatic necrosis after hepatocyte transplantation. Wild type hepatocytes were isolated and transplanted through the spleen into the liver of recipients. The recipients were divided into three groups. Group 1 received wild hepatocyte transplantation only; group 2 received acute CCl4, (1.2 ml/kg mouse body weight) 24 h after wild hepatocyte transplantation; group 3 received acute CCl4 (1.2 ml/kg mouse body weight) 24 h after p27 null hepatocyte transplantation, followed by weekly injections of CCl4 (0.5 ml/kg mouse body weight) for 4 or 8 weeks. Recipients were sacrificed 1 week after the final treatment with CCl4 or vegetable oil only. Liver segments were flash-frozen for histology and DPPIV staining to quantitate donor cell engraftment and proliferation. Even though the repopulation was less than 1%, after 4 weeks, mice receiving either acute insult only (group 2, 0.34 ± 0.10%) or acute insult combined with chronic liver injury (group 3, 0.53 ± 0.23%) had significant liver repopulation than mice absence of a liver regenerative stimulus (group 1, 0.15 ± 0.08%,P = 0.03, and P =0.01, respectively). However, after 8 weeks, the differences became insignificant (group 2, 0.20 ± 0.16% and group 3, 0.46 ± 0.36% versus group 1, 0.05 ± 0.03%; P = 0.09, and P =0.18, respectively). This indicated that liver injury increased the initial engraftment of transplanted hepatocytes but did not augment the proliferation of transplanted hepatocytes.
Then, we transplanted cells capable of higher proliferation activity to evaluate the proliferation of this particular cells in the diseased liver. p27Kipl is a cyclin-dependent kinase inhibitor that stably interacts with cyclin A and cyclin E/Cdk complexes to negatively regulate cell proliferation. p27 null mice are about 25% larger than wild-type littermates and show hyperplasia and organomegaly of multiple organs, including the liver. Hepatocytes in the p27 null liver show accelerated entry into S phase after partial hepatectomy, and p27 inactivation also synergizes with the inactivation of another kinase inhibitor, pi 8, to stimulate hepatocyte proliferation after partial hepatectomy. These results are consistent with previous studies by Karnezis et al., who demonstrated increased [3H]thymidine and BrdU incorporation into DNA in primary hepatocytes from p27 null mice compared with eild hepatocytes.
Because p27 null mice and DPPIV null mice are on different genetic backgrounds, these studies also required development of an immunotolerant DPPIV null mouse capable of accepting allogeneic hepatocytes. This was accomplished by breeding DPPIV null mice with Rag2 null mice, which are deficient in T and B cells due to inability in VDJ rearrangement, and are capable of accepting both allogeneic and xenogeneic cells. DPPIV-/- mice were crossed with Rag2-/- mice to produce heterozygous mice for both DPPIV and Rag2 gene deletions. The double heterozygous mice were mated together to produce either DPPIV-/-Rag2+/- or DPPIV+/- Rag2-/- mice, which were then crossed with each other to generate DPPIV-/- /Rag2-/- double knockout mice. Fig. 12 shows that DPPIV-/- /Rag2-/- mice accepted hepatocytes from various rodent species, including 129sv x C57B1/6 mice, 129sv x CD-1 mice, Balb c mice, and Sprague-Dawley rats, and the transplanted cells can also be identified by DPPIV histochemical staining.
To compare the study that transplanted wild hepatocyte in conjunction with partial hepatectomy, we transplanted P27-/- null hepatocytes into DPPIV-/-/Rag2-/- mice in conjunction with two thirds partial hepatectomy at the time of cell transplantation and followed proliferation of transplanted cells for 6 months (Fig. 13). In contrast to cessation of proliferation by wild hepatocytes in DPPIV-/- mice after 1 month (Fig. 13A), there was a modest increase in the size of DPPIV+/+ cell clusters at 2-3 months after P27+/+ hepatocyte transplantation (Fig. 13B, C), and although infrequent, larger clusters were clearly evident at 6 months after cell transplantation (Fig. 13D).
Thereafter, we attempts to increase the proportion of transplanted hepatocytes by stimulating liver regeneration through repeated CCl4-induced hepatic necrosis after hepatocyte transplantation. For convenience to compare the groups transplanted with p27 null hepatocytes and wild hepatocytes, we divided the mice into three groups and defined as: group 4, received p27 null hepatocyte transplantation only; group 5, received acute CCl4, (1.2 ml/kg mouse body weight) 24 h after p27 null hepatocyte transplantation; and group 6, received acute CCl4 (1.2 ml/kg mouse body weight) 24 h after p27 null hepatocyte transplantation, followed by weekly injections of CCl4 (0.5 ml/kg mouse body weight) for 4 or 8 weeks. Liver segments were also flash-frozen for histology and DPPIV staining to quantitate donor cell engraftment and proliferation.
We found that when an acute liver regenerative stimulus was included at the time of cell transplantation, the P27-/- null hepatocytes repopulated and formed larger clusters than hepatocytes without any regeneration stimulus after 4 weeks (group 5, 0.48 ± 0.23% versus group 4, 0.18 ± 0.17%) and 8 weeks (group 5, 0.40 ± 0.25% versus group 4, 0.10 ± 0.13%), although the differences were small and not statistically significant (P = 0.08, and P =0.15, respectively). However, transplanted cells proliferated and repopulated significantly better in liver with acute liver insult and chronic liver injury than in a normal quiescent liver after 4 weeks (group 6, 1.27 ± 0.76% versus group 4, 0.18 ± 0.17%; P = 0.03), and even better after 8 weeks (group 6, 3.27 ± 2.06% versus group 4, 0.10 ± 0.13%,P = 0.01). In addition, in groups with acute liver insult, P27-/- null hepatocytes repopulated better in those with chronic liver injury after 8 weeks (group 6, 3.27 ± 2.06% versus group 5, 0.40 ± 0.25%,P=0.02) than those with acute insult only, and in mice received acute insult with chronic liver injury, the repopulation of liver increased significantly from 4 weeks to 8 weeks(1.27 ± 0.76%versus 3.27 ± 2.06%, P=0.049).
Further comparison was made between transplantation of p27-/- null and wild hepatocytes. After four weekly doses of CCl4 (Fig. 14), there was no significant increase in the number or size of DPPIV+/+ clusters in livers of mice transplanted with wild hepatocytes (Table 9, Fig. 14A). In contrast, however, after four weekly doses of CCl4, the livers of mice transplanted with p27-/- null hepatocytes showed an increase in both the number and size of DPPIV4 clusters (Table 9. Fig. 14C), and the total number of cells in 100x field increased by two-fold, although the data did not reach statistical significance because of variations between individual animals (22.13 ± 9.00 versus10.27 ± 2.31, P = 0.075). Four additional treatments with CCl4 (a total of eight weekly doses) markedly increased the number and size of DPPIV4 clusters produced by p27 null hepatocytes (Table 9, Fig. 14D), but no such enhancement was observed with p27 wt hepatocytes (Table 9, Fig. 14B). These differences were clearly statistically significant (35.58 ± 16.10 versus 7.53 ± 5.56 in 100x field, P = 0.015).
To quantitate the proliferation of transplanted cells after repeated liver injury, 20 random fields at l00x magnification were examined from multiple sections of each lobe of the liver to determine the average number of clusters per field and the average number of cells per cluster. As showed in Fig. 15, even though the majority of clusters contained one or two cells with both p27 null and wt hepatocytes, the percentage of clusters containing three or more cells was greater with p27 null hepatocytes after four weekly treatments with CCl4 (Fig. 15A). The difference in cluster size between p27 null and wild hepatocytes became much more dramatic after eight weekly treatments with CCl4. Under these conditions, the largest clusters (four in total) derived from wild hepatocytes contained 6-10 cells and none contained more than 10 cells (Fig. 15B). Under comparable circumstances with p27 null hepatocytes, 268 clusters contained 6-10 cells and 110 clusters contained more than 10 cells (Fig. 15B). In addition, the largest clusters derived from p27 null hepatocytes contained 40-50 cells compared with 6-10 cells with p27 wt hepatocytes (cf. Fig. 14D and B). Assuming that each cluster is spherical and derived from a single transplanted cell, the largest clusters with p27 null hepatocytes represent an average of eight cell divisions, whereas those from p27 wt hepatocytes represent an average of five to six cell divisions. These results demonstrate that transplanted p27 null hepatocytes exhibit augmented proliferative activity compared with transplanted wild hepatocytes in the normal host liver and this difference is cumulative during the regenerative response to repeated liver injury.
Liver repopulation by transplanted hepatocytes was measured too. With transplanted hepatocytes alone (quiescent liver), there was no difference in the percent repopulation between p27 null and wt hepatocytes, either at 4 or 8 weeks after cell transplantation. Addition of a single acute liver regenerative stimulus slightly increased liver repopulation by transplanted cells, but again there was no statistically significant difference between p27 null and wild hepatocytes. However, weekly repeated small doses of CCl4 augmented liver repopulation by p27 null hepatocytes versus wild hepatocytes (Fig. 16). After four weekly cycles of CCl4 administration, the extent of liver repopulation by p27 null hepatocytes was more than double that observed with wt hepatocytes, but the difference was still not statistically significant (P=0.14). After eight weekly cycles of CC4 administration, there was a sevenfold greater liver repopulation by p27 null hepatocytes (3.27%, range 1.2-6.1%) compared with wild hepatocytes (0.46%, range 0.04-0.63%) that was highly statistically significant (p= 0.036). In selected areas of high liver repopulation after 8 weeks of CC l4 treatment (predominantly in the periportal and midzonal regions), p27 null hepatocytes replaced up to 12-15% of total hepatic mass compared with 1-1.5% with wild hepatocytes. These results all indicated that p27 null hepatocyte still under normal control in regenerated liver and can not proliferate spontaneously and indefinitely after liver regeneration finished. On the other hand, chronic liver injury enhanced the proliferation capacity of the p27 null hepatocytes, resulted in augmentation of liver repopulation.
Section Two: Chronic Liver Injury before Cell Transplantation Attenuate Function of Hepatic Kupffer cell and Enhanced Liver Repopulation
Last but not least, we want to find out the effect of preceding chronic liver disease on the proliferation capacity of transplanted cells. In the mean time, we developed an animal model using acetaminophen, a very common analgesic, to induce acute liver injury because acetaminophen poisoning is very common in human. In the UK 50% of poisoning admissions involve acetaminophen. In the United State, acetaminophen intoxication accounts for nearer 10% poisoning admissions and that resulted in acute liver failure in as many as 800 people, and one-third of whom died every year. The major pathways of acetaminophen in liver include glucuronidation or sulphation, and the resulting non-toxic conjugates excreted by the kidney. In addition, another pathway involves the cytochrome P-450 (CYP) system, especially CYP2E1, by which acetaminophen is metabolized to the highly reactive metabolite N-acetyl-p- benzoquinoneimine, that may bind covalently with hepatic proteins causing cellular necrosis. The toxic effect of N-acetylp-benzoquinoneimine can be eliminated by the natural antidote glutathione. When taken overdose, acetaminophen will cause a potentially fatal, hepatic centrilobular necrosis. Acute liver failure induced by acetaminophen is a multisystem disorder, with acute renal failure, hypotension, sepsis, coagulopathy, encephalopathy and cerebral edema. Patients who present within 24 h of an acetaminophen overdose can be managed medically. But for those who developed acute liver failure, meticulous supportive care and transplantation is considered for those patients who will die without liver transplantation.
In the fourth portion, we transplanted wild hepatocytes into acetaminophen intoxicated mice without (group 1) or with (group 2) repeated CCl4 injury twice a week for 4 weeks before cell transplantation, and then to sacrifice the mice 7 and 14 days after the transplantation to evaluate the repopulation capacity of transplanted cells.
We found that after chronic repeated liver regenerative stimulus before cell transplantation, number and cluster number of the transplanted cell in 100x field were larger than those without repeated regenerative stimulus at 7 days (21.53 ± 6.29 versus 8.25 ± 3.98, and 14.86 ± 4.62 versus 6.59 ± 3.39; P<0.001 and P<0.001, respectively; Fig. 17, Table 10). The cell number and cluster number remained larger significantly in group with repeated CCl4 injury at 14 days too (17.29 ± 4.40 versus 10.16 ± 3.31, 12.59 ± 3.19 versus 7.90 ± 1.58; P=0.001, and P=0.004, respectively, Table 10), and therefore better hepatocyte repopulation (0.44% versus 0.16% at 7 days, and 0.35% versus 0.20% at 14 days). However, the cell number, the cluster number and repopulation decreased from 7 days to 14 days in liver with repeated CCl4 injury.
Even though the percentage of clusters containing three or more cells was greater in liver with repeated chronic injury at 7 days (P=0.007), the majority of clusters still contained only one or two cells, and the percentage of cluster containing three or more cells were similar 14 days after transplantation. This indicated that repeated chronic liver injury increased engraftment and stimulated initial proliferation of transplanted cells but the proliferation ability is limited after hepatocyte transplantation without further stimulation.
After cell transplantation, the transplanted cell deposited in the hepatic sinusoid, and the Kupffer cells are activated shortly. Therefore, a significant fraction of transplanted cells were destroyed especially when the transplanted cells are entrapped in portal spaces. It’s also reported that perturbation of Kupffer cells activity might increase engraftment of transplanted cell. To elucidate the role of hepatic macrophage on the engraftment of transplanted cell, we use fluorescence immunohistochemical staining to assess the distribution, and use lipopolysaccharide (LPS) stimulation to evaluate the activity of the hepatic macrophage. The hepatic macrophage distributed scatter throughout the plate in quiescent liver. After acute acetaminophen intoxication, the number of macrophage increased but still exhibited generalized distribution. After repeated chronic liver injury, the hepatic macrophages increased and were distributed prominently in the portal area, and even high up after additional acetaminophen insult (Fig. 18). Before LPS stimulation, the TNF-α mRNA producing ability of the Kupffer cells isolated from mice treated with acute on chronic liver injury was prominent than those from acute injury only (TNF-αmRNA/GAPDH level, 117.8 ± 12.3 versus 73.9 ± 12.4, P=0.012). But after LPS stimulation, macrophages isolated from mice that treated with acute on chronic liver injury had the TNF-α mRNA producing ability significantly lower than those from mice receiving acute acetaminophen only (TNF-αmRNA/GAPDH level, 175.7 ± 54.6 versus 465.6 ± 54.2, P=0.004: Fig. 20). It indicated that chronic repeated liver injury before cell transplantation attenuated the function of hepatic Kupffer cells, and might contribute to increased engraftment of transplanted cells. But the molecular relationship between transplanted cell engraftment and Kupffer cell function deserved further evaluation.
In this study, we demonstrated that REG1A, PAP and LAP18 were all correlated with cell proliferation and tumor invasiveness in hepatocellular carcinoma. We showed that both REG1A and PAP were correlated well with β-catenin mutation, but HCCs with PAP expression alone had the highest frequencies of well differentiated and low-stage tumors, the least frequent early tumor recurrence, and the best 5-year survival, while HCC coexpressed with REG1A is associated with more advanced disease and may contribute to tumor progression. In addition, we showed that LAP18 overexpression was associated with bigger tumor, high-grade, high-stage HCC with vascular invasion, more frequent early tumor recurrence, and worse prognosis. These findings suggest that LAP18 confers growth advantage and facilitates tumor cell growth and invasion capacity. These observations provide novel in vivo evidence of PAP, REG1A and LAP18 on cell proliferation capacity in hepatocellular carcinoma, and might offer specific targets for therapeutic strategies concerning cell proliferation in hepatocellular carcinoma.
Furthermore, we demonstrated augmented hepatocyte transplantation with genetic alternated hepatocytes in vivo. Our results revealed that, p27 null hepatocytes did not differ from their wild counterpart in their engraftment or proliferative activity in quiescent livers, and exhibited a slightly proliferative advantage under an acute proliferative stimulus. This suggests that once the liver mass has returned to normal, the p27 null hepatocytes cannot overcome the normal growth control, but the genetic altered hepatocytes can proliferate significantly after repeated liver regeneration stimulus. However, uncontrolled proliferation often impairs cellular differentiation and can pose unacceptable tumor risk. Therefore, a delicate balance should be achieved that would augment cellular proliferation and benefit transplanted hepatocytes, but would not increase tumor risk by the transplanted cells.
Finally, we demonstrate that chronic liver disease increased engraftment of transplanted cells and this phenomenon might result from attenuation of resident Kupffer cells in the recipient liver. This indicated that instead of doing harm for the patient, chronic liver disease might be beneficial for transplanted cells to be engrafted and repopulated in the liver. Additional studies to attenuate the function of hepatic Kupffer cells and increase the repopulation of transplanted cells are mandatory in the near future.
Subjects
肝細胞癌
肝細胞移植
細胞增殖
hepatocellular carcinoma
hepatocyte transplantion
cell proliferation
SDGs
Type
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