A potent DNA synthesis inhibitor expressed by the immortal cell line SUSM-1

We have previously reported the production of DNA synthesis inhibitor proteins by both quiescent and senescent human diploid fibroblasts. Young, proliferating fibroblasts do not produce such inhibitors, but are capable of responding to either the quiescent or senescent cell DNA synthesis inhibitors. Recently, we have analyzed the immortal cell line SUSM-1 (derived from normal liver fibroblasts following exposure to carcinogen) for inhibitory activity. We have found that SUSM-1 cells produce a factor capable of inhibiting DNA synthesis in young fibroblasts. Crude extracts prepared from SUSM-1 cells inhibit DNA synthesis in a dose-dependent manner at concentrations 10-fold lower than those of either senescent or quiescent fibroblast cell extracts. SUSM-1 cells are incapable of responding to the inhibitor they produce, as are three other immortal human cell lines tested. One immortal cell line, HeLa, does respond to the SUSM-1 inhibitor, though to a lesser degree than observed with normal young fibroblasts. One hypothesis is that the DNA synthesis inhibitor protein(s) of senescent cells plays a role in determining the finite in vitro life span of normal cells. The results reported here suggest that SUSM-1 cells may have escaped senescence through loss of a receptor or cofactor for the inhibitor protein(s).     

RecA-like activity in mammalian cell extracts of different origin

The occurrence of a RecA-like activity similar to the one detected in the fibroblast cell line GM1492 derived from a patient suffering from the autosomal recessive disease Bloom's syndrome has been investigated in cell extracts of different origin. The formation of D-loop containing joint molecules from phi X174 RFI DNA and fragments of phi X174 single-stranded DNA by partially purified extracts was measured by a filter binding assay. The RecA-like activity, dependent on ATP and Mg2+, was detected at an elevated level only in the human and rodent cell lines, GM1492 and CHO respectively. The level of activity in DNA-cellulose-purified cell extracts from these cell lines was 4-7-fold higher compared to normal human fibroblasts. Low levels of activity were also detected in extracts from two additional Bloom's syndrome fibroblast cell lines, Fanconi's anemia fibroblasts, virus- (Epstein-Barr virus, Simian virus 40) transformed human cells and human placenta. Cell extracts from rat testis, spleen and calf thymus were also of low activity.     

Homologous plasmid recombination is elevated in immortally transformed cells.

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.     

Clonal heterogeneity in telomerase activity and telomere length in tumor-derived cell lines

The ribonucleoprotein, telomerase, is responsible for the maintenance of telomere length in most immortal and cancer cells. Telomerase appears to be a marker of human malignancy with at least 85% of human cancers expressing its activity. In the present study, we examined a series of tumor-derived and in vitro immortalized cell lines for telomerase activity levels, telomere lengths, and expression levels of the RNA and catalytic components of telomerase. We found significant variability in both telomere lengths and telomerase activity in clones from tumor cells. In addition, the levels of telomerase components or telomerase activity were not predictive of telomere length. Data from clonally derived cells suggest that critically shortened telomeres in these tumor-derived cell lines may signal activation of telomerase activity through an increase in the expression of the catalytic subunit of telomerase. Although clones with low telomerase shorten their telomeres over time, their subclones all have high levels of telomerase activity with no telomere shortening. In addition, analysis of early clones for telomerase activity indicates substantial variability, which suggests that activity levels fluctuate in individual cells. Our data imply that cell populations exhibit a cyclic expression of telomerase activity, which may be partially regulated by telomere shortening.     

Chromosome 7 suppresses indefinite division of nontumorigenic immortalized human fibroblast cell lines KMST-6 and SUSM-1.

Using nontumorigenic immortalized human cell lines KMST-6 (KMST) and SUSM-1 (SUSM), we attempted to identify the chromosome that carries a putative senescence-related gene(s). These cell lines are the only ones that have been established independently from normal human diploid fibroblasts following in vitro mutagenesis. We first examined restriction fragment length polymorphisms on each chromosome of these immortalized cell lines and their parental cell lines and found specific chromosomal alterations common to these cell lines (a loss of heterozygosity in KMST and a deletion in SUSM) on the long arm of chromosome 7. In addition to these, we also found that introduction of chromosome 7 into these cell lines by means of microcell fusion resulted in the cessation of cell division, giving rise to cells resembling cells in senescence. Introduction of other chromosomes, such as chromosomes 1 and 11, on which losses of heterozygosity were also detected in one of the cell lines (KMST), to either KMST or SUSM cells or of chromosome 7 to several tumor-derived cell lines had no effect on their division potential. These results strongly suggest that a gene(s) affecting limited-division potential or senescence of normal human fibroblasts is located on chromosome 7, probably at the long arm of the chromosome, representing the first case in which a specific chromosome reverses the immortal phenotype of otherwise normal human cell lines.     

Immortalization in a normal foreskin fibroblast culture following transduction of cyclin A2 or cdk1 genes in retroviral vectors

Human diploid fibroblasts (HDF) rarely, if ever, undergo spontaneous transformation to an immortalized cell type. Here we report the immortalization of an HDF cell line following transduction with cyclin A2 or cdk1 human genes via retroviral vectors. Fluorescence in situ hybridization (FISH) studies using the retroviral vector as a probe indicate that these cell lines are monoclonal. No telomerase activity could be detected in these cell lines, and the telomere length in the immortalized cells was observed to be 10-20 kb longer than that in low-passage cells from the parental fibroblast line. Cytogenetic studies revealed that the immortal lines share common chromosomal aberrations. FISH studies with a probe for p53 revealed loss of one copy of this gene which was associated with reduced steady-state levels of both p53 and p53-regulated p21(WAF1/Sdi1/CIP1) messages in both quiescent and proliferating immortalized cultures relative to the parental cells. Additional FISH studies with probes for p16(INK4a) and Rb, carried out after the immortalized cells proliferated in excess of 100 population doublings, also revealed loss of one copy of these genes in both cell lines. These cell lines, together with the well-characterized parental cells, could provide useful research material for the study of the mechanisms of immortalization and of regulation of proliferative senescence in HDF.     

Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts

Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization. © 1996 Wiley-Liss, Inc.     

Intermolecular plasmid recombination in fibroblasts from humans with DNA damage-processing defects

We have evaluated the ability of immortalized human fibroblasts to recombine transfected plasmid DNA. A number of cell lines from normal individuals and from patients with DNA damage-processing defects were examined. Two plasmid recombination substrates were derived from pSV2neo and contained nonoverlapping deletions in the aminoglycoside phosphotransferase II gene. Intermolecular recombination was assessed by two methods after cotransfection. In a short-term, extrachromosomal recombination assay, low molecular weight DNA was extracted from the human cells 48 h after transfection, and recombinant plasmids were detected by transformation into appropriate indicator bacteria. In a long-term stable recombination assay the fibroblasts were cotransfected and G418-resistant colonies allowed to form. By the former assay all but two cultures were recombination-proficient, whereas all were recombination-proficient by the latter assay. The efficiency of transfection of human cells with plasmids appears to be a major variable affecting recombination. Recombination can be stimulated by uv irradiation of plasmid DNA prior to transfection. Cells from patients with Fanconi anemia, ataxia telangiectasia, and xeroderma pigmentosum complementation groups A, C, D, E, and G are not defective at intermolecular plasmid recombination.     

A monoclonal antibody identifies a 215 000-dalton nuclear envelope protein restricted to certain cell types

The monoclonal antibody, AGF2.3, was isolated from mice immunised with the human promyeloid cell line HL60. By immunofluorescence and immunoelectron microscopy the antibody was shown to bind to the nuclear envelope in uninduced HL60 cells. Immunofluorescent staining was reduced to very low levels in HL60 cells induced to mature to monocytes or neutrophils by addition of 12-0-tetradecanoylphorbol-13-acetate or dimethyl sulfoxide respectively. Blood neutrophils did not express the antigen. Weak immunofluorescent staining of cell nuclei was observed in peripheral blood lymphocytes and in sections of normal human kidney, tonsil and skin epithelium. The AGF2.3 antigen was strongly expressed on the nuclei of 21/21 haemopoietic cell lines and 21/25 permanent non-haemopoietic cell lines representing various cell types. In contrast, the antigen was not expressed by any of six primary (untransformed) cell cultures. These included fibroblasts, endothelial cells and keratinocytes. The antigen was expressed in the Q10 SV-40 transformed cell line derived from a non-expressing primary fibroblast culture. AGF2.3 antibody precipitated a protein with an apparent subunit molecular weight of approximately 215 kDa from Triton X-100 extracts of HL60 and HeLa cells labelled with 35S-methionine. This protein was not detectable in extracts of primary skin fibroblasts prepared in parallel. We conclude that AGF2.3 antibody recognises a previously undescribed protein associated with the nuclear envelope which is expressed at high levels in most transformed cell lines but which is weakly expressed or absent in normal tissues and primary cell cultures.     

Analysis of the capacity of extracts from normal human young and senescent fibroblasts to support DNA synthesis in vitro

Cytoplasmic extracts from early-passage (young), late-passage (senescent) normal human fibroblast (HF) cultures and immortalized human cell lines (HeLa, HT-1080, and MANCA) were analyzed for their ability to support semiconservative DNA synthesis in an in vitro SV40-ori DNA replication system. Unsupplemented extracts from the three permanent cell lines were demonstrated to be active in this system; whereas young HF extracts were observed to be minimally active, and no activity could be detected in the senescent HF extracts. The activity of these extracts was compared after supplementation with three recombinant human replication factors: (1) the catalytic subunit of DNA polymerase alpha (DNA pol-alpha-cat), (2) the three subunits of replication protein A (RPA), and (3) DNA topoisomerase I (Topo I). The addition of all three recombinant proteins is required for optimum activity in the young and senescent HF extracts; the order of the level of activity is: transformed > young HF > senescent HF. Young HF extracts supplemented with RPA alone are able to support significant replicative activity but not senescent extracts which require both RPA and DNA pol-alpha-cat for any detectable activity. The necessary requirement for these factors is confirmed by the failure of unsupplemented young and senescent extracts to activate MANCA extracts that have been immunodepleted of DNA pol-alpha-cat or RPA. Immunocytochemical studies revealed that RPA, DNA pol-alpha, PCNA, and topo I levels are higher in the immortal cell types used in these studies. In the HF cells, levels of DNA pol-alpha-cat and PCNA are higher (per mg protein) in the low-passage than in the senescent cells. By contrast, RPA levels, as determined by immunocytochemical or Western blot studies, were observed to be similar in both young and senescent cell nuclei. Taken together, these results indicate that the low to undetectable activity of young HF extracts in this system is due mainly to reduced intracellular levels of RPA, while the senescent HF extracts are relatively deficient in DNA polymerase alpha and probably some other essential replication factors, as well as RPA. Moreover, the retention of RPA in the senescent HF nuclei contributes to the low level of this factor in the cytoplasmic extracts from these cells.     

Mouse embryonic stem cells exhibit high levels of extrachromosomal homologous recombination in a chloramphenicol acetyltransferase assay system.

Mouse embryonic stem (ES) cells were compared to COS1 and CV1 cells for their ability to perform extrachromosomal homologous recombination. RSVCAT plasmid substrates consisting of overlapping chloramphenicol acetyltransferase (CAT) gene fragments were transiently transfected into cells and extracts were assayed for CAT activity. Approximately 10% activity, relative to transfection with a complete CAT gene, was recovered for the recombination substrates in each of the cell lines tested. ES cells, therefore, as other cell lines, are capable of high levels of extrachromosomal recombination.     

Relationship between contact inhibition and intranuclear S100C of normal human fibroblasts

Many lines of evidence indicate that neoplastic transformation of cells occurs by a multistep process. For neoplastic transformation of normal human cells, they must be first immortalized and then be converted into neoplastic cells. It is well known that the immortalization is a critical step for the neoplastic transformation of cells and that the immortal phenotype is recessive. Thus, we investigated proteins downregulated in immortalized cells by two-dimensional gel electrophoresis. As a result, S100C, a Ca(2+)-binding protein, was dramatically downregulated in immortalized human fibroblasts compared with their normal counterparts. When the cells reached confluence, S100C was phosphorylated on threonine 10. Then the phosphorylated S100C moved to and accumulated in the nuclei of normal cells, whereas in immortalized cells it was not phosphorylated and remained in the cytoplasm. Microinjection of the anti-S100C antibody into normal confluent quiescent cells induced DNA synthesis. Furthermore, when exogenous S100C was compelled to localize in the nuclei of HeLa cells, their DNA synthesis was remarkably inhibited with increase in cyclin-dependent kinase inhibitors such as p16(Ink4a) and p21(Waf1). These data indicate the possible involvement of nuclear S100C in the contact inhibition of cell growth.     

Multi-step neoplastic transformation of normal human fibroblasts by Co-60 gamma rays and Ha-ras oncogenes

As reported previously (Namba et al., 1985; Namba, 1985), normal human fibroblasts were transformed into immortal cells with abnormal karyotypes by Co-60 gamma-ray irradiation. These immortally transformed cells (KMST-6) showed no clonability in soft agar and were not tumorigenic. However, by treatment with Ha-ras oncogenes derived from a human lung carcinoma or Harvey murine sarcoma virus, the KMST-6 cells acquired elevated clonability in soft agar and transplantability in nude mice. All the tumors produced grew progressively without showing regression and killed the mice. The tumors were also serially transplantable into other mice. The Ha-ras oncogene alone did not convert normal human fibroblasts into either immortal or tumorigenic cells. Our current data suggest that gamma rays worked as an initiator of carcinogenesis in normal human cells, giving rise to chromosome aberrations and immortality, and the Ha-ras oncogene played a role in the progression of the immortally transformed cell population to a neoplastic one showing enhanced colony formation in soft agar and tumorigenicity in nude mice.     

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.     

Telomere loss: mitotic clock or genetic time bomb?

The Holy Grail of gerontologists investigating cellular senescence is the mechanism responsible for the finite proliferative capacity of somatic cells. In 1973, Olovnikov proposed that cells lose a small amount of DNA following each round of replication due to the inability of DNA polymerase to fully replicate chromosome ends (telomeres) and that eventually a critical deletion causes cell death. Recent observations showing that telomeres of human somatic cells act as a mitotic clock, shortening with age both in vitro and in vivo in a replication dependent manner, support this theory's premise. In addition, since telomeres stabilize chromosome ends against recombination, their loss could explain the increased frequency of dicentric chromosomes observed in late passage (senescent) fibroblasts and provide a checkpoint for regulated cell cycle exit. Sperm telomeres are longer than somatic telomeres and are maintained with age, suggesting that germ line cells may express telomerase, the ribonucleoprotein enzyme known to maintain telomere length in immortal unicellular eukaryotes. As predicted, telomerase activity has been found in immortal, transformed human cells and tumour cell lines, but not in normal somatic cells. Telomerase activation may be a late, obligate event in immortalization since many transformed cells and tumour tissues have critically short telomeres. Thus, telomere length and telomerase activity appear to be markers of the replicative history and proliferative potential of cells; the intriguing possibility remains that telomere loss is a genetic time bomb and hence causally involved in cell senescence and immortalization.     

Down-regulation of T-STAR, a growth inhibitory protein, after SV40-mediated immortalization.

Normal human cells can undergo a limited number of divisions, whereas transformed cells may have an extended life span and can give rise to immortal cells. To isolate genes involved in the immortalization process, gene expression in SV40-transformed preimmortal human fibroblasts was compared with expression in SV40-transformed immortalized fibroblasts using an mRNA differential display. We found that the growth-inhibitory protein testis-signal transduction and activation of RNA (T-STAR) a homologue of cell-cycle regulator Sam68, is strongly down-regulated in immortalized cells. Overexpression of T-STAR in the SV40-transformed immortalized cells resulted in a strong reduction of colony formation, whereas deletion of the RNA-binding domain of T-STAR abrogated this effect. Down-regulation of testis-signal transduction and activation of RNA (T-STAR) expression is found only in immortal cells isolated after a proliferative crisis accompanied with massive cell death. The strict correlation of down-regulation of T-STAR expression only in those immortal cells that arose after a clear proliferative crisis suggests that the loss of T-STAR might be necessary to bypass crisis.     

Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.     

Correlation between complementation group for immortality and DNA synthesis inhibitors

Previous studies had demonstrated that a DNA synthesis inhibitor(s) was produced by senescent but not young human diploid fibroblasts (HDF). Analysis of immortal human cell lines led to the finding that SUSM-1, carcinogen-treated immortal human liver fibroblast cells, expressed a potent inhibitor of DNA synthesis that was active in proliferation-competent young HDF but did not affect the SUSM-1 cell line itself. To determine whether one mechanism of escape from senescence to the immortal phenotype involved the loss of response to such DNA synthesis inhibitors, we initiated the present study analyzing a larger number of immortal human cell lines representative of the four complementation groups for indefinite division identified to date. We have found a correlation between the assignment of a cell line to Complementation Group D and the production of DNA synthesis inhibitors coupled with inability to respond to the inhibitory factors. We have also observed a correlation between the ability of immortal cell lines to respond to such DNA synthesis inhibitory factors and assignment to Complementation Group B. These data suggest DNA synthesis inhibitors are involved in the limited lifespan of normal cells and that the immortalization process may involve alterations in the activity of or response to such inhibitors.