Cytogenetic analysis of SV40-transformed human breast epithelial cells

The SV40-transformed breast epithelial cell lines established by Chang et al. [1] were shown to be hypotetraploid and characterized by six chromosome markers: M1 i(1q), M2 del(1)(q21), M3 i(6p), M4 del(1)(q11), M5 t(8p;12q), and M6 dir dup(11)(p12→pter). The presence of common chromosome markers indicates that these cell lines are probably derived from the same original transformed cell.     

Characterization of defective SV40 isolated from SV40-transformed cells.

Defective SV40 viruses were isolated from SV40-transformed monkey, human and hamster cells after Sendai virus-mediated fusion of the transformed cells with TC7 cells, a stable line of African green monkey kidney cells. Viral isolates were concentrated and purified and the defective viruses examined by electron microscopy. The buoyant densities in CsCl of the defective viruses ranged between 1.32 and 1.33 g/cc. DNA isolated from defective viruses was characterized by dye-buoyant density centrifugation and by velocity sedimentation in neutral CsCl. The DNA was heterogeneous in size and contained some covalently closed double-stranded circular molecules.     

Transformation of mouse mammary epithelial cells by papovavirus SV40

Primary and established murine mammary epithelial cells and wild-type SV40 were employed to study the phenomenon of epithelial cell transformation. Thirteen independent transformed cell lines were derived. All contained SV40 intranuclear T antigen. Eight transformed mammary cell lines were examined ultrastructurally and all were found to exhibit pronounced epithelial cell characteristics, including desmosomes and tight junctions. Growth studies revealed that while normal mammary cells were unable to grow in low serum (2% FBS), established Cl S1 mammary cells and SV40-transformed mammary epithelial cells replicated well. Cell densities achieved by the transformants were only slightly elevated in high serum (13% FBS) over normal cell values. All the transformants formed colonies on plastic and exhibited anchorage-independent growth in methylcellulose. Five of the transformed lines were tumorigenic in syngeneic animals, in marked contrast to the lack of transplantability usually observed with SV40-transformed mouse fibroblasts. Anchorage-independent growth was not a predictor of tumorigenic potential in this system. The transformants exhibited a spectrum of responsiveness to exogenous growth factors. This study establishes that the SV40-murine mammary cell system is a valid model for analyses of the process and consequences of epithelial cell transformation, in general, and mammary cell transformation in particular.     

Expression of receptors for enterotoxigenic Escherichia coli during enterocytic differentiation of human polarized intestinal epithelial cells in culture.

To study the expression of human intestinal receptors for enterotoxigenic Escherichia coli (ETEC), the human polarized intestinal epithelial cell line Caco-2 in culture and several subpopulations of HT-29 cells in culture--parental (mainly undifferentiated) HT-29 cells (HT-29 Std), an enterocytelike subpopulation obtained by selection through glucose deprivation (HT-29 Glc-), and an enterocytelike subpopulation obtained by selection through glucose deprivation which maintains its differentiation characteristics when switched back to standard glucose-containing medium (HT-29 Glc-/+)--were used. Since Caco-2 spontaneously differentiated in culture under standard culture conditions (in the presence of glucose) and HT-29 cells were undifferentiated when cultured under standard conditions (HT-29 Std) and differentiated when grown in a glucose-free medium (HT-29 Glc-), we studied the expression of the receptors for colonization factor antigens (CFA) I, II, and III and the 2230 antigen of ETEC in relation to enterocytic differentiation. We provide evidence that expression of ETEC CFA receptors develops in parallel with other differentiation functions of the cultured cells. The expression of ETEC-specific brush border receptors was studied by indirect immunofluorescence using antibodies raised against purified ETEC CFA. No ETEC receptors were detected in HT-29 Std or short-term-cultured Caco-2 cells. However, among the population of HT-29 Std cells, 2 to 4% of the cells were found to bind ETEC, and these cells expressed positive carcinoembryonic antigen immunoreactivity. This indicated that among the population of undifferentiated HT-29 cells, clusters of differentiated cells were present. ETEC CFA receptors were expressed in the apical and basolateral domains of differentiated HT-29 cells, whereas in differentiated Caco-2 cells only apical expression was observed. Both in HT-29 cells (HT-29 Glc-/+) and in Caco-2 cells cultured under standard conditions, ETEC CFA receptors develop as a function of day in culture. This indicated that the expression of the ETEC CFA receptors was a growth-related event. Indeed, ETEC CFA receptors developed in step with the apical expression of differentiation-associated proteins.     

Detergent-resistant membrane microdomains and apical sorting of GPI-anchored proteins in polarized epithelial cells

Detergent-insoluble microdomains or rafts play a crucial role in many cellular functions: membrane traffic, cell signalling and human diseases. In this work we investigate the role of rafts in the sorting of GPI-anchored proteins in polarized epithelial cells. In contrast to MDCK cells, the majority of endogenous GPI-anchored proteins are sorted to the basolateral surface of Fischer rat thyroid cells (Zurzolo et al., J. Cell Biol. 121, 1031-1039, 1993). We analyzed a set of transfected GPI proteins in order to understand the role of the GPI anchor and of association with rafts for apical sorting. We found that the GPI moiety is necessary but not sufficient for apical sorting of GPI proteins and that the ectodomain has a major role. We propose a new model in which the stabilization of proteins into rafts, probably mediated by interactions between protein ectodomains and a putative receptor, plays a crucial role in apical sorting.     

BKV and SV40 infection of human kidney tubular epithelial cells in vitro

The interaction of BKV with its natural target cells, human kidney epithelial cells, has not been studied. In vitro infections of human primary kidney epithelial cells were performed to investigate a BKV infection in its natural host cell. BKV undergoes a lytic replication cycle in this system: high levels of T antigen expression were first detected at 36 h postinfection, while viral DNA replication, capsid protein expression, and progeny virus were observed at 48 h postinfection. It was observed that the related polyomavirus SV40 is incapable of infecting human kidney epithelium except in the presence of the GM1 ganglioside, recently reported to be an SV40 receptor.     

SV40-transformed cells express SV40 T antigen-related antigens on the cell surface

SV40 tumor antigen (T-Ag)-related antigens were detected serologically on the surface (surface T) of living SV40-transformed human and mouse monolayer cells by an ¹²⁵I-protein A binding assay. In immunofluorescence analysis, these cells were negative for surface T. However, on mKSA, a SV40-transformed mouse cell line grown in suspension or on SV40-transformed human and mouse monolayer cells put into suspension, surface T could be visualized by immunofluorescence microscopy. The antisera used in these experiments were raised in rabbits with purified, SDS-denatured SV40 T-Ag or came from hamsters bearing SV40 tumors. Both types of antisera had in common high titers against SV40 T-Ag (?1:1000). All these antisera were negative on normal cells or on polyoma virus-transformed cells. The specificity of both antisera for SV40 T-Ag-related binding sites on the surface of SV40-transformed cells were demonstrated by an ¹²⁵I-IgG blocking assay in which preincubation of the cells with rabbit anti-T-Ag serum inhibited the binding of hamster SV40 tumor serum to the cell surface by about 85%. These results demonstrate the expression of T-Ag-related antigens on the surface of living cells and, therefore, support the hypothesis that SV40 T-Ag-related antigens participate in the formation of the SV40-specific tumor transplantation antigen (TSTA).     

Selective immortalization of mammary epithelial cells by microinjection of SV40 DNA into human milk cells

Immortalization of mammary epithelial cells has been obtained by microinjection of SV40 DNA into epithelial cells from primary cultures of rabbit mammary glands. A modification of this technique, allowing selective immortalization of a specific cell type of mammary epithelial cells from human milk by microinjection of SV40 DNA, is described. It includes the selection of luminal epithelial cells from human milk cells after identification of these cells by antibodies directed against specific markers such as intermediate filaments and membrane antigens, and finally, the introduction of SV40 DNA into cell nuclei which will selectively immortalize this restricted cell population. This procedure can be used to derive permanent cell lines with a defined phenotype when only a few cells are available within a heterogeneous cell population.     

Expression of cell adhesion complexes in epithelial cells seeded on biomaterial surfaces

Clinical studies indicate that soft tissue responses around dental implants vary, depending on the material used. It is therefore also possible that there are differences in how epithelial cells attach to various biomaterial surfaces. We studied the adhesion of cultured epithelial cells to five different dental material surfaces and to glass. The efficacy of adhesion was evaluated by using scanning electron microscopy (SEM) and immunofluorescence microscopy (IF) with antibodies to vinculin and alpha(6)beta(4) integrin, two cell surface molecules that are functional in epithelial cell adhesion. Our results indicate that epithelial cells adhere and spread more avidly on metallic surfaces (titanium, Ti(6)Al(4)V titanium alloy, dental gold alloy) than on ceramic surfaces (dental porcelain, aluminum oxide). As revealed by SEM, cells on metallic surfaces had a flattened morphology and formed multicellular islands. On porcelain and aluminum oxide most cells were round and adhesion occurred as single cells. Surface coverage was over twofold on metallic surfaces as compared to ceramic surfaces. IF of cells grown on metallic surfaces revealed vinculin in well-organized focal contacts and alpha(6)beta(4) integrin in punctate patterns typical of prehemidesmosomes. On porcelain and aluminum oxide surfaces the cells were mostly round and showed less well-organized adhesion complexes. Our results indicate that smooth metallic biomaterial surfaces are optimal for epithelial cell adhesion and spreading. These findings may have clinical implications in the design of transgingival implant structures.     

Expression of SV40 T antigen in finite life-span hybrids of normal and SV40-transformed fibroblasts

The fusion of normal human fibroblasts with SV40-transformed human fibroblasts resulted in hybrid clones, 85% of which exhibited a finite in vitro life-span. Foci of rapidly dividing cells appeared in 15% of the hybrid clones. The cells within these foci repopulated the culture and could then be subcultured through more than 100 population doublings. One or two foci of dividing cells occurred per culture of 10⁵ or more cells. The change to an indefinite life-span was, therefore, a rare event. All hybrid clones, including those that exhibited a finite in vitro life-span, expressed viral T antigen. Thus, even though viral DNA was present and being expressed in all hybrid clones, the senescent phenotype was dominant in these hybrids.This work is in partial fulfillment of requirements for a doctoral degree.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IV. Inhibition by human interferon of expression of SV40 TrAg in SV40-infected monkey cells.

Human interferon inhibits the synthesis of SV40 transplantation rejection antigen (TrAg) in SV40-infected but not in SV40-transformed monkey cells. The synthesis of SV40 tumor antigen as detected by the indirect immunofluorescence test and the large and small T antigens as detected by the immunoprecipitation with sera from tumor-bearing hamsters and electrophoresis in SDS-gels was similarly affected in SV40-infected monkey cells. These results suggest that the induction of SV40-specific TrAg in the cytolytic cycle depends upon a viral, rather than a host, message.     

Protein metabolism in SV40-infected cells

Simian virus 40-induced alterations in protein metabolism were investigated in confluent monolayers of BS-C-1 monkey kidney cells. Viral-induced changes were observed which were specific for both the prereplicative and the replicative phases of infection.The prereplicative phase is that period of the infection prior to the onset of viral DNA synthesis, which begins at 19–20 hr. A viral-induced stimulation of protein synthesis is first observable at about 10–12 hr, and by 20 hr after infection the rate of protein synthesis has increased to more than 20% above that of mock-infected cells.Gel electrophoresis of the proteins synthesized during the prereplicative period did not reveal any differences from control cells in any cell fraction except the cytosol, i.e., the nonparticulate portion of the cytoplasm. In this fraction several new species were observed, but together they did not constitute more than 1% of the overall rate of protein synthesis. An inhibition of labeling of one, and possibly two, host proteins was also discerned in the cytosol. Each of the polypeptide changes just described was also observed in infected cells which had been treated with cytosine arabinoside. This confirms the assignment of these effects to the early phase of infection, as it has been well-established that cytosine arabinoside blocks viral DNA synthesis and all late viral functions, but has little effect on early events.During the replicative period the rate of protein synthesis continues to increase to more than 50% above that of mock-infected cells. Starting at 20 hr postinfection, proteins destined for the nucleus are synthesized at higher relative rates than those of cytoplasmic fractions. As a consequence, a very large progressive accumulation of protein in the nucleus begins at about 40 hr after infection. Much of this increased nuclear mass could be accounted for as progeny virus particles found in the membranous fraction of sonicated nuclei.Gel electrophoresis of newly-made proteins late in infection showed that the two major viral structural proteins constituted about 20% of cellular protein synthesis. There was an additional viral-induced nonstructural polypeptide in the cytosol which constituted less than 1% of the newly-made proteins. As expected, these three new proteins were not synthesized in infected cells which had been treated with cytosine arabinoside. It was also observed that the labeling of the major host protein in the nuclear membrane was inhibited during the late phase of infection.     

Association of SV40 large tumor antigen and cellular proteins on the surface of SV40-transformed mouse cells

A cellular protein with a molecular weight of about 53,000 (53K) and histocompatibility antigens (mouse H-2 antigens) have been reported to be associated with viral-specified proteins in transformed cells. We investigated whether such associations could be detected on the surface of SV40-transformed mouse cells. A differential immunoprecipitation technique was adapted so that surface-associated antigens could be detected independently from intracellular antigens. Cells grown as monolayers were enzymatically labeled with ¹²⁵I-Na using a lactoperoxidase-catalyzed reaction, or metabolically labeled with either [³⁵S]methionine or ³²P_i, and were then incubated with antisera against mouse H-2 antigens or SV40 large T-antigen (T-ag) or with monoclonal antibodies against mouse 53K nonviral T-antigen (nvT-ag). The cells were then disrupted with an NP40 solution, the extracts were clarified by centrifugation, and the immune complexes in the supernatant fluids adsorbed with protein A-containing Staphylococcus aureus. Internal antigens, present in the cell lysates, were precipitated by a second incubation with antiserum and the antigen-antibody complexes collected again with immunoadsorbent. The precipitated proteins were eluted and analyzed by SDS-polyacrylamide gel electrophoresis. Reconstruction experiments established that T-ag released from the nucleus during the extraction procedure was not combining with free antigen-binding sites on antibodies bound to the cell surface in the external reaction, that nuclear unbound T-ag was not exchanging with bound surface antigen during extraction, and that the surface reaction was not due to nuclear T-ag released from dead cells and nonspecifically adsorbed onto the surface of living cells. Iodinated 94K T-ag was specifically immunoprecipitated by T antibody during the external reaction; an iodinated 53K polypeptide was coprecipitated. Conversely, labeled T-ag and 53K were coprecipitated from surface-iodinated transformed cells by monoclonal antibodies against mouse 53K nvT-ag. Thus, it appears that SV40 large T-ag and cellular 53K protein are associated on the surface as well as within SV40-transformed mouse cells. In contrast, no detergent-stable complex between T-ag and mouse H-2 antigens was detected on the transformed cells. The possibility that molecular interactions between viral- and cell-coded proteins could be involved in determining some of the observed transformation-related cellular phenotypic changes is discussed.     

DNA replication in SV40-infected cells. IX. The inhibition of a gap-filling step during discontinuous synthesis of SV40 DNA

SV40 DNA apparently replicates by a discontinuous mechanism similar to that proposed for bacteria and bacteriophages. Transient, 4–5 S oligonucleotide intermediates are formed during a brief pulse-labeling with ${}^3$H-thymidine, and these fragments are rapidly joined to preexisting progeny strands (Fareed and Salzman, 1972). An inhibitor of DNA synthesis, hydroxyurea, interferes with the joining of these short 4–5 S oligonucleotide intermediates into longer DNA molecules. This results in the accumulation of replicating forms of SV40 DNA with 4–5 S oligonucleotide fragments hydrogen-bonded at the replication forks to parental DNA strands.Replicating molecules of SV40 DNA isolated from lytically infected cells treated with hydroxyurea were repaired in an in vitro DNA synthesizing system using T4 DNA polymerase and Escherichia coli polynucleotide ligase. The experimental results indicate that at least 80% of the short 4–5 S fragments are separated from themselves or longer progeny molecules by single-stranded gaps in the DNA double helix. These gaps can be repaired in vitro, and the fragments ligased to form longer polynucleotide chains.A model for SV40 DNA replication involving two different DNA polymerases is discussed.