Establishment of cell lines for quality control of prenatal genetic diagnosis by gene transfection / 浙江大学学报·医学版

OBJECTIVE@#To establish a cell lines for quality control of prenatal genetic diagnosis.@*METHODS@#The recombined SV40LTag-pcDNA3.1(-) vector was constructed and transfected by lipidosome into human amniotic fluid cells with common aneuploidy. Positive clones were screened by G418, and the immortality of transfected cell line was identified.@*RESULTS@#Cell line with karyotype of 46, XY, t(8;19)(q24.3;q13.1) from primary amniotic fluid cells was established. Karyotype analytical results indicated that the cell line at its 15th generation maintained the same karyotype of its primary cell.@*CONCLUSIONS@#Gene can lead to immortality of amniotic fluid cells, which contributes to preparing cell lines for internal and external quality control in prenatal genetic diagnosis.

Bone marrow-derived mesenchymal stem cells migrate to healthy and damaged salivary glands following stem cell infusion / 国际口腔科学杂志·英文版

Xerostomia is a severe side effect of radiation therapy in head and neck cancer patients. To date, no satisfactory treatment option has been established. Because mesenchymal stem cells (MSCs) have been identified as a potential treatment modality, we aimed to evaluate stem cell distribution following intravenous and intraglandular injections using a surgical model of salivary gland damage and to analyse the effects of MSC injections on the recruitment of immune cells. The submandibular gland ducts of rats were surgically ligated. Syngeneic adult MSCs were isolated, immortalised by simian virus 40 (SV40) large T antigen and characterized by flow cytometry. MSCs were injected intravenously and intraglandularly. After 1, 3 and 7 days, the organs of interest were analysed for stem cell recruitment. Inflammation was analysed by immunohistochemical staining. We were able to demonstrate that, after intravenous injection, MSCs were recruited to normal and damaged submandibular glands on days 1, 3 and 7. Unexpectedly, stem cells were recruited to ligated and non-ligated glands in a comparable manner. After intraglandular injection of MSCs into ligated glands, the presence of MSCs, leucocytes and macrophages was enhanced, compared to intravenous injection of stem cells. Our data suggest that injected MSCs were retained within the inflamed glands, could become activated and subsequently recruited leucocytes to the sites of tissue damage.

U6 is unsuitable for normalization of serum miRNA levels in patients with sepsis or liver fibrosis

MicroRNA (miRNA) levels in serum have recently emerged as potential novel biomarkers for various diseases. miRNAs are routinely measured by standard quantitative real-time PCR (qPCR); however, the high sensitivity of qPCR demands appropriate normalization to correct for nonbiological variation. Presently, RNU6B (U6) is used for data normalization of circulating miRNAs in many studies. However, it was suggested that serum levels of U6 themselves might differ between individuals. Therefore, no consensus has been reached on the best normalization strategy in 'circulating miRNA'. We analyzed U6 levels as well as levels of spiked-in SV40-RNA in sera of 44 healthy volunteers, 203 intensive care unit patients and 64 patients with liver fibrosis. Levels of U6 demonstrated a high variability in sera of healthy donors, patients with critical illness and liver fibrosis. This high variability could also be confirmed in sera of mice after the cecal ligation and puncture procedure. Most importantly, levels of circulating U6 were significantly upregulated in sera of patients with critical illness and sepsis compared with controls and correlated with established markers of inflammation. In patients with liver fibrosis, U6 levels were significantly downregulated. In contrast, levels of spiked-in SV40 displayed a significantly higher stability both in human cohorts (healthy, critical illness, liver fibrosis) and in mice. Thus, we conclude that U6 levels in the serum are dysregulated in a disease-specific manner. Therefore, U6 should not be used for data normalization of circulating miRNAs in inflammatory diseases and previous studies using this approach should be interpreted with caution. Further studies are warranted to identify specific regulatory processes of U6 levels in sepsis and liver fibrosis.

Identification and characterization of two critical sequences in SV40PolyA that activate the green fluorescent protein reporter gene

Alu repeats or Line-1-ORF2 (ORF2) inhibit expression of the green fluorescent protein (GFP) gene when inserted downstream of this gene in the vector pEGFP-C1. In this work, we studied cis-acting elements that eliminated the repression of GFP gene expression induced by Alu and ORF2 and sequence characteristics of these elements. We found that sense and antisense PolyA of simian virus 40 (SV40PolyA, 240 bp) eliminated the repression of GFP gene expression when inserted between the GFP gene and the Alu (283 bp) repeats or ORF2 (3825 bp) in pAlu14 (14 tandem Alu repeats were inserted downstream of the GFP gene in the vector pEGFP-C1) or pORF2. Antisense SV40PolyA (PolyAas) induced stronger gene expression than its sense orientation (PolyA). Of four 60-bp segments of PolyAas (1F1R, 2F2R, 3F3R and 4F4R) inserted independently into pAlu14, only two (2F2R and 3F3R) eliminated the inhibition of GFP gene expression induced by Alu repeats. Deletion analysis revealed that a 17 nucleotide AT repeat (17ntAT; 5'-AAAAAAATGCTTTATTT-3') in 2F2R and the fragment 3F38d9 (5'-ATAAACAAGTTAACAACA ACAATTGCATT-3') in 3F3R were critical sequences for activating the GFP gene. Sequence and structural analyses showed that 17ntAT and 3F38d9 included imperfect palindromes and may form a variety of unstable stem-loops. We suggest that the presence of imperfect palindromes and unstable stem-loops in DNA enhancer elements plays an important role in GFP gene activation.

The absence of JC virus antigens in Indian children with medulloblastomas.

BACKGROUND: The human polyoma virus, also known as the JC virus (JCV), replicates predominantly in the oligodendrocytes, the myelin producing cells in the central nervous system and results in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) especially in immunosuppressed patients with AIDS. Several investigators have also documented the presence of the viral genome and early and late antigens in a variety of brain tumors particularly in medulloblastomas, gliomas and ependymomas. Reports also indicate the presence of JCV in patients with colon cancer. The T antigen of JCV has been postulated to have oncogenic potential as substantiated by animal experiments. Although JCV infects 80% of the population, there are scant epidemiological studies regarding JCV from India. There are also reports of the low prevalence of PML in patients with AIDS from India and Africa. AIM: This study was undertaken to investigate if Indian children with medulloblastomas also show evidence of JCV. METHODS: Twenty-two consecutive cases of medulloblastomas were investigated for the presence of T antigen and agnoprotein of JCV in biopsy specimens by immunohistochemistry. Antibodies to the agnoprotein antigen raised in rabbits and a monoclonal antibody against SV40 T antigen raised in mice that cross-reacts with JCV T antigen were used. RESULTS: Out of 22 patients, 4 had desmoplastic tumors while the rest had classical tumors. All children were below the age of 10. Results indicate that while PML tissues showed consistent immunostaining both with antibody to T antigen and agnoprotein antibody, none of the tumors showed any positive staining for JC viral antigens. CONCLUSION: JCV antigens could not be detected by immunohistochemistry in the tumor tissues of Indian children with medulloblastomas.

Detection of SV40 Large T Antigen in Malignant Lymphomas

BACKGROUND: The association of simian virus 40 (SV40) with certain types of human cancers, including malignant lymphomas, has been a topic of interest for some time. Although the virus is distributed worldwide, its incidences vary according to the specific types of tumors, and the epidemiological areas. The aim of this study was to investigate the frequency of SV40 in malignant lymphomas among Korean patients. METHODS: One hundred seventy three cases of malignant lymphomas were evaluated by immunohistochemical staining for SV40 large T antigen (TAg), using an extremely sensitive, tyramide based, catalyzed signal amplification method. RESULTS: From 158 non-Hodgkin's lymphomas, including 115 diffuse large B-cell lymphomas, and 15 Hodgkin's lymphomas, none of the cases were positive for SV40 TAg. CONCLUSIONS: SV40 does not appear to be related to the pathogenesis of malignant lymphomas among Koreans.

Immortalization of rat epiphysis cartilage cells induced by simian virus 40 large T antigen gene transfection / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To establish immortalized epiphysis cartilage cell strains in order to provide a stable cell resource for cell substitution and gene therapies of growth retardation.</p><p><b>METHODS</b>Plasmid pEGFP-IRES2-SV40LTag containing simian virus 40 large T antigen gene was transfected into primarily cultured epiphysis cartilage cells of the newborn rat using the lipofectin transfection method. Colonies were isolated by G418 selection and cultured to immortalized cell strains. Fibroblast growth factor receptor-3 (FGFR-3), anti-collagen type II and type X antibodies were used to identify cultured cells and to investigate the capability of differentiation of the transfected cells. SV40LTag expression in expanded cell strains was identified by RT-PCR, Southern blot and immunocytochemistry method.</p><p><b>RESULTS</b>Anti-G418 cell clone was obtained, which was confirmed as FGFR-3 positive epiphysis cartilage cells with the capability of stable proliferation. mRNA and protein of SV40LTag were expressed in transfected cells after stable transfection. The transfected cells were expanded to immortalized cell strains and named as immortalized epiphysis cartilage cells. The immortalized cells were elliptic or triangular, with two or three short axons. The immortalized epiphysis cartilage cell strains had stable biological characters.</p><p><b>CONCLUSIONS</b>SV40LTag gene transfection can immortalize epiphysis cartilage cells. The establishment of FGFR-3 positive immortalized epiphysis cartilage cell strains may provide a stable cell resource for cell substitution and gene therapies of growth retardation.</p>

Expression of SV40Tag, Rb and IRS-1 in glioma detected by tissue microarray and their relation with tumorigenesis and progression of gliomas / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To determine the expression of SV40Tag, Rb and IRS-1 in gliomas and to identify their function in gliomagenesis and progression.</p><p><b>METHODS</b>Tissue microarrays were constructed containing 118 samples including human glioma and meningioma, experimental glioma, and normal human brain tissue. The expression of SV40Tag, Rb, IRS-1, SV40Tag combined with Rb, and SV40Tag combined with IRS-1 were assayed by immunofluorescence or immunohistochemical techniques. The expression ratio and level were analyzed.</p><p><b>RESULTS</b>The expressions of SV40Tag, Rb and IRS-1 were detected in gliomas and benign brain tumors. Their positive expression rate in glioma was 65.9%, 64.6% and 48.8%, respectively, with a statistically non-significant difference between the malignant and benign brain tumors. The malignant degree was positively correlated with SV40Tag and IRS-1, but negatively correlated with Rb expression. The combined expression rate of SV40Tag and Rb was 51.2%, and the combined expression rate of SV40Tag and IRS-1 was 40.2%. In the normal human brain tissue only the expression of Rb (77.8%, 7/9) and IRS-1 (22.2%, 2/9) were detected, but expression of SV40Tag could not be observed.</p><p><b>CONCLUSION</b>Our findings that no expression of SV40Tag was observed in normal human brain tissue indicates that expression of SV40Tag may play an important role in the pathogenesis of glioma. It may be assumed that after SV40 virus invading human body, Rb disfunction and IRS-1 activation promote the malignant transformation of cells, which could be one of important factors in pathogenesis and procession of glioms.</p>

The establishment of conditional MT transgenic mice model / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To construct the doxycycline-inducible MT transgenic mice model, and provide a basis for the study of hemangioma as well as MT molecular function in vivo.</p><p><b>METHODS</b>Tetracycline-controlled expression systems were employed to this study. A conditional transgenic vector combining the two transcriptional units on a single plasmid was constructed, and the MT gene was subcloned into this vector. To minimize any potential interference, the two elements were spaced with a 1.2 kb cHS4 insulator. To shield the transgene from the affection of chromosomal position effect and improve its expression efficiency, another cHS4 insulator was inserted into the upstream of transgene cassette. After transient transfection of cells in vitro, and analyzing the relative quantification of MT transcripts (target) in mRNA samples by semi-quantitative RT-PCR method, the pronuclear microinjection technique was used to introduce the purified transgene into the chromosomes of fertilized mice eggs, in order to obtain transgenic positive animals. The MT expression in positive mouse was induced through adding deoxycycline in drinking water. Phenotype analysis was done by pathology, and MT expression was confirmed by RT-PCR.</p><p><b>RESULTS</b>The conditional transgenic vector was constructed successfully, and the expression of MT in vitro was regulated by doxycycline. Five transgenic positive mice were obtained through pronuclear microinjection. After MT induction, one transgenic mice developed hemangiomas, and the expression of MT was confirmed by RT-PCR method. The others were active and in breeding.</p><p><b>CONCLUSION</b>Conditional MT transgenic animal model was constructed successfully, and may provide platform for the experimental research of hemangioma as well as the MT molecular function in vivo.</p>

Characterization of a brain tumor cell line established from transgenic mice expressing the vasopressin SV-40 T antigen

We previously reported that transgenic mice produced with a transgene consisting of the SV40 T antigen and vasopressin without the 3'-flanking region exhibit brain tumors and lymphoma. In this study, transgenic mice were produced with the fusion gene containing the SV40 T antigen and the whole vasopressin gene with the 3'-flanking region. Six transgenic mice were generated, five which died after 2-6 weeks. The remaining founder mouse was investigated for fusion gene expression and tumor progression at the age of 6 weeks. Brain tumor cells were characterized for phenotypes and transgene expression. During in vitro cell cultures, the phenotypic appearances at 10, 20, and 30 passages were as a uniform monolayer with similar growth rates. The site of SV40 T antigen integration was in the A2 region of chromosome 11, and SV40 T antigen was expressed at the same level in cells of both earlier and later passages. Thirty passages were probably insufficient to reach crisis and immortalization. These cells enriched brain tumor cell compositions with astrocytes and neuronal cells.

Immortalization of embryonic fibroblasts in heat shock transcription factor 1 knockout mouse / 中南大学学报(医学版)

OBJECTIVE@#To establish immortalized embryonic fibroblast lines in heat shock transcription factor 1 (HSF1) HSF1-/- and HSF1+/+ mice and to provide experimental models to study the function of HSF1.@*METHODS@#A mammalian expression vector (pSV3neo) containing the SV40 large T antigen was used to transfect the HSF1-/- and HSF1+/+ mouse embryonic fibroblast using Lipofectamine 2000. Colonies were screened by G418 and expanded to immortalized cell lines. PCR was used to detect the integration of the large T antigen with genome in the mouse embryonic fibroblast. Expression of SV40 large T antigen gene in expanded cells was identified by RT-PCR. HSP70 expression was examined by Western blot in the embryonic fibroblast lines.@*RESULTS@#The stable growth and serial propagation were observed in the HSF1-/- and HSF1+/+ cell lines for six months. The mRNA of SV40 T antigen gene expressed in the two cell lines. HSP70 expression could not be induced in the heat-treated HSF1-/- mouse embryo fibroblasts.@*CONCLUSION@#The immortalized cells of HSF1+/+ and HSF1-/- mouse embryo fibroblasts are successfully established.

Immortalization of human umbilical vein endothelial cells by transfected with hTERT and SV40LT / 中华心血管病杂志

<p><b>OBJECTIVE</b>To immortalize human umbilical vein endothelial cells (HUVECs) by ectopic expression of the telomerase reverse transcriptase enzyme (hTERT), and by Simian Virus 40 Large T (SV40LT) antigen without malignant transformation.</p><p><b>METHODS</b>Two different retroviruses that contained hTERT/SV40LT cDNA fragment and drug resistance gene were constructed, and were used to transfect normal primary HUVECs. The transfected cells were screened with 500 microg/ml G418 and 4 microg/ml puromycin. Drug resistance cell clones were selected 3 days after transfection and cultured for further studies. An under inverted microscope and a scanning electron microscope were used to observe the morphology and growth of the cells. The expression of VIII factor and transfected DNA fragments were detected for identification of the endothelial origin and successful transfection. And the expression of E-selectin and endothelial lipase with or without the stimulus of TNF-alpha were also assayed to analyze the biological activity of the transfected cells.</p><p><b>RESULTS</b>The cells were homogenous, closely apposed, large, flat, and polygonal, displayed a characteristic ovoid nucleus with one or two nucleoli and formed monolayer with polygonal shape without overlapping. Immunocytochemical staining showed the existence of VIII factor. SV40LT/hTERT antigen expressed by the transfected cells was detected, while the contrasts had non-expression. Telomerase activity of the cell was detected in the transfected cells, which was 0.36 at 12 th passage and 0.38 at 50 th passage. However, the activity in the normal HUVECs was 1.12 at the first passage and 0.06 at the third passage assayed by PCR-ELISA. Both E-selectin and endothelial lipase were all specific in endothelial cells. The expressions of these two were also detected. And the expression of E-selectin can be up-regulated with the stimulus of TNF-alpha, while the expression of endothelial lipase was not unregulated significantly.</p><p><b>CONCLUSION</b>Ectopic expression of hTERT and SV40LT can effectively immortalize HUVECs without tumorigenesis.</p>

Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen.</p><p><b>METHODS</b>Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination.</p><p><b>RESULTS</b>The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor, hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-alpha (tumor necrosis factor-alpha), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found.</p><p><b>CONCLUSION</b>Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.</p>

The formation of transgenic mice with hemangiomas / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To set up the animal model of hemangioma by microinjecting the PyMT transgenic DNA.</p><p><b>METHODS</b>Constructing the transgenic PyMT gene, and microinjecting it into fertilized embryos which were transferred to pseudopregnant recipients then. Observing the phenotype of the newborn-mice, detecting the integration of transgenic DNA by PCR, and analyzing the histological morphon of the neoplasm of the mice.</p><p><b>RESULTS</b>The transgenic DNA was proved to be right and has been microinjected into 579 fertilized embryos, 62 mice were born. Within the 62 mice, one mouse was found being the phenotype of hemangioma. PyMT gene was expressed in the total DNA of the transgenic mouse by PCR.</p><p><b>CONCLUSION</b>It could be a good way to build animal model of hemangioma with transgenic PyMT DNA.</p>

Establishment of immortalized ameloblastoma cell line TAM-1 / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To establish an immortalized ameloblastoma cell line.</p><p><b>METHODS</b>The primary cultured ameloblastoma cells were transfected with pRSV-Tag using Transfect AMINE kit. Tansfected cells were passaged to pass through crisis period and immortalize.</p><p><b>RESULTS</b>Cultured ameloblastoma cells were composed predominantly of closely packed small polygonal cells with epithelial morphology. They had limited life-span of 51 days in vitro. The small polygonal cells were eventually replaced by large flattened cells and subsequently became senescent and dead. On the other side, those tumor cells transfected with SV40Tag could live for a longer time. The majority of them died in crisis period while the survived cells from crisis period gained the ability to proliferate. There was no morphological change in TAM-1 compared with original cultured cells. A cell clone was harvested which was alive and keeping on proliferating after having been subcultured for 25 times. It was named TAM-1. The epithelial origin of TAM-1 was confirmed by strong immunoreactivity for cytokeratin in contrast to negative vimentin expression. It was detected that SV40Tag had been transfected into TAM-1 genesome and expressed continuously by PCR and RT-PCR.</p><p><b>CONCLUSIONS</b>TAM-1 is immortalized ameloblastoma cell line in vitro.</p>

A study on the morphological characters of immortalized mandibular condylar chondrocyte / 华西口腔医学杂志

<p><b>OBJECTIVE</b>The purpose of this study was to compare the morphological character between immortalized mandibular condylar chondrocyte (IMCC) and primarily cultured mandibular condylar chondrocyte (MCC).</p><p><b>METHODS</b>The phase contrast microscope, photomicroscope and transmission electron microscope were used to observe the morphological character of IMCC and MCC. The highresolution pathological image and word report system-1000 (HPIAS-1000) was used to compare the size of IMCC and MCC.</p><p><b>RESULTS</b>The phase contrast micrography showed that MCCs in primary culture underwent distinct morphological changes with respect to shape, size, and density of the cells. The majority of MCCs were in polygonal shape earlier in culture, while more fusi-form and spindle-shaped cells were found after 4-5 passages. While IMCCs were polygonal-shaped, similar to MCCs. Subculture, freezing and recovering had no effect on cellular shape of IMCC. Transmission electron microscopy indicated that MCC had chondrocyte-like phenotype, while IMCC looked like prechondroblast or immature chondrocyte. Some of IMCCs had irregular nucleus, and the proportion of nucleus/cytoplasm changed. By analysis of HPIAS-1000, the diameter and area of IMCC were obvious smaller than those of MCC (P < 0.01).</p><p><b>CONCLUSION</b>IMCC retain the main morphological character of MCC, and also keep a stable phenotype, which belong to immature chondrocytes, similar to cells in the proliferative zone.</p>

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.

Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage

The rat hepatocytes were immortalized using a temperature-sensitive mutant of SV40 large T antigen (tsT) to develop as a possible substitute for primary hepatocytes. Four rat hepatocyte lines that have been developed and maintained more than passage 50, were characterized for their cellular morphology, T antigen and p53 expression, chromosomes, liver-specific differentiation, telomerase activity and anchorage independent growth. All of four cell lines showed a typical epithelial cell morphology, but the population-doubling time became short with passage: 18 to 60%. T antigen expression was increased with passage about 3 to 65 times at permissive temperature but decreased significantly at non-permissive temperature. The expression level of p53 unchanged during passages was also decreased at non-permissive temperature. The distribution of chromosome number changed somewhat with passage. The production levels of albumin and urea in four cell lines were 2.4 to 13.0% and 7.5 to 19.9% of those produced in primary hepatocytes, respectively and were decreased to an undetectable level with passage. Telomerase activity was increased 10 fold following immortalization of cells, but anchorage independent growth of cells did not develop. These results indicate that conditionally immortalized hepatocytes become dedifferentiated with in vitro passage, which may be caused by marked chromosomal damages that occur with compulsive and continuous replications by the increment of T antigen content with passage and its sequential inhibition of p53 function.

Mechanisms of cell transformation induced by polyomavirus

Polyomavirus is a DNA tumor virus that induces a variety of tumors in mice. Its genome encodes three proteins, namely large T (LT), middle T (MT), and small T (ST) antigens, that have been implicated in cell transformation and tumorigenesis. LT is associated with cell immortalization, whereas MT plays an essential role in cell transformation by binding to and activating several cytoplasmic proteins that participate in growth factor-induced mitogenic signal transduction to the nucleus. The use of different MT mutants has led to the identification of MT-binding proteins as well as analysis of their importance during cell transformation. Studying the molecular mechanisms of cell transformation by MT has contributed to a better understanding of cell cycle regulation and growth control