Isolation of novel cDNA transformation markers from SV40-transformed human keratinocytes

Differential screening of a cDNA library was used to isolate probes for mRNAs that are induced in simian virus 40 (SV40)-transformed human keratinocytes. Several of these cDNAs hybrid select mRNAs which encode transformation-induced proteins found in the cytoskeletal component of SV40-transformed keratinocytes. One of these cDNAs was used to study the phenotype of normal and transformed cell lines derived from various tissues. We found that mRNA encoding the novel transformation-induced proteins is expressed in two squamous carcinoma cell lines derived from the oral epithelium, four SV40-transformed keratinocyte cell lines, and two SV40-transformed fibroblasts. Normal or transformed lymphoid cells or cell lines derived from carcinoma of the cervix do not express mRNAs which hybridize to these probes. The results from this study suggest that these probes may be used to detect markers of transformation in certain cell types.     

Fusion-induced differentiation of SV40-infected human keratinocytes

Cells in cultures of human keratinocytes infected by the oncogenic virus, SV40, were fused by exposure to polyethylene glycol (PEG). Fusion induced differentiation of the infected cells within 1–2 days, as measured by staining with orange G/acid fuchsin and by the production of cornified envelopes. The extent of induced differentiation was found to be correlated with the degree of multinucleation. The inductive effect of fusion was manifest during the early serial passages post-infection, but was eventually lost as the keratinocytes became increasingly transformed over time.     

SV40 early region oncoproteins and human cell transformation

We now understand neoplastic transformation to be the consequence of multiple acquired genetic alterations. The combination of these acquired changes confer the various phenotypes that constitute the clinical features of cancer. Although only rare human cancers derive from a viral etiology, the study of DNA tumor viruses that transform rodent and human cells has led to a greater understanding of the molecular events that program the malignant state. In particular, investigation of the viral oncoproteins specified by the Simian Virus 40 Early Region (SV40 ER) has revealed critical host cell pathways, whose perturbation play an essential role in the experimental transformation of mammalian cells. Recent work has re-investigated the roles of two SV40 ER oncoproteins, the large T antigen (LT) and the small t antigen (ST), in human cell transformation. Co-expression of these two oncoproteins, together with the telomerase catalytic subunit, hTERT, and an oncogenic version of the H-Ras oncoprotein, suffices to transform human cells. LT inactivates two key tumor suppressor pathways by binding to the retinoblastoma protein (pRB) and p53. The ability of ST to transform human cells requires interactions with PP2A, an abundant family of serine-threonine phosphatases. Here we review recent developments in our understanding of how these two viral oncoproteins facilitate human cell transformation.     

Re-expression of differentiated properties in SV40-infected human epidermal keratinocytes induced by 5-azacytidine

Infection of human epidermal keratinocytes by the oncogenic virus SV40 leads to progressive inhibition of the normal differentiation process in vitro. Treatment of infected cells with 5-azacytidine (5-aza-CR) over a 24-h period produced a striking enlargement and pronounced flattening of cells within 5-7 days following removal of the agent. This morphological change was accompanied by a several-fold increase in the number of cells staining positively for the cell envelope precursor protein, involucrin, and in the exfoliation of cornified envelope bearing cells from the monolayer. The drug-treated cultures at high passage levels were stained by immunofluorescence using monoclonal antibodies to keratin classes associated with different epidermal layers. These experiments revealed that 5-aza-CR caused the re-expression of two keratin classes (suprabasal and stratum corneum-associated), whose synthesis had been suppressed during the transformation process. 5-Aza-CR also brought about re-expression of 58 and 56 kD keratin markers of epithelial keratinization and stratification, as well as of 40 and 49-52 kD keratin markers of viral transformation. However, the responsiveness to the drug was gradually lost over time following infection.     

Patterns of cell communication and differentiation in SV40 transformed human keratinocytes

Fluorescein dye microinjection was used to demonstrate changes in communication between human epidermal keratinocytes grown in vitro after infection by the oncogenic virus, SV40. Whereas keratinocytes are normally fully coupled to each other, dye spread becomes progressively restricted to small cell subpopulations after infection, although dye coupling is increased when the infected cells attain high densities. Reduction or enhancement of dye coupling is correlated with similar changes in the extent of cytochemical differentiation and cornified envelope formation.     

Analysis of stable and unstable viral forms in SV40-infected human keratinocytes

We analyzed the state of the genomic DNA of the papovavirus SV40 in human keratinocytes as viral-infected cells gradually acquired a transformed phenotype over time. Initially, the vast majority of the viral DNA is maintained either in a full-length supercoiled form or as truncated subgenomic fragments with little evidence of integration. However, analyses of clonal populations revealed great heterogeneity and instability of the viral DNA, and we were able to isolate one clonal subpopulation in which integrated forms of the virus appeared to predominate. Similarly, uncloned populations eventually ceased production of the "free" viral DNA after several years in culture and instead came to display tandemly repeated SV40 copies at a single host integration site. Interestingly, Bg1 II digestion of host DNA generated restriction fragments containing the integrated SV40 DNA, which were of differing sizes in cultures at the 144th vs the 163rd serial passage suggesting modification or rearrangement of sequences at or near the integration site. Host sequences flanking the integrated viral DNA at the 163rd serial passage have been isolated on restriction fragments generated by Eco RI, Bam HI, and Hpa II digestion. These analyses suggest that the integrated virus is linearized near the Bg1 I site and contains a large deletion in the SV40 early region at one of the viral-host junctions.     

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells.

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.     

Temperature sensitive cells in the study of SV40 lysis versus SV40 transformation

Temperature sensitive variant clones of African green monkey kidney cell line (BSC-1) have been isolated which were transformed at a high frequency by SV40 at the restricted temperature, but were lytic to SV40 infection at the permissive temperature. Loss of contact inhibition and cell proliferation at the restricted temperature appeared to be in some way related to the synthesis of T antigen in these variant cell lines.     

Fluorescence spectroscopy of normal, SV40-transformed human keratinocytes, and carcinoma cells

Native fluorescence emission and excitation spectra of SV40 infected human keratinocytes, A431 and SCC324 carcinoma cells, and normal human keratinocytes were measured and compared. A difference in the intracellular metabolic state of NADH was found between the normal cells and the cancer or virus-transformed cells. The observed difference, namely an increased proportion of bound, mitochondrial NADH in the cancer and virus-infected cells, manifests as a blue spectral shift in the emission spectra.     

p53 and transformation by SV40

The large T antigen of SV40 is able to immortalize and transform primary and established cells in culture, and can, at least in certain cases, confer a tumorigenic phenotype on the infected cell. T antigen has been shown to induce cellular DNA synthesis in the infected cell and this activity is likely to be instrumental in T antigen mediated oncogenesis. A property of T antigen which may be of paramount importance to its oncogenic and mitogenic activities is its ability to specifically bind and stabilize the cellular protein p53. p53 has been implicated in the control of the passage of the cell from G0 arrest to G1 and S phase. Furthermore, altered p53 expression is strongly associated with various phenotypes of the transformed state, and p53 has been identified as an immortalizing oncogene. Thus it is possible that p53-fixation by T antigen is responsible for its transforming potential. In this article, the transforming activities of T antigen and p53 are reviewed, and the possible relevance of p53-binding to T antigen-induced transformation is discussed.     

Altered patterns of keratin synthesis in human epidermal keratinocytes transformed by SV40

Transformation of human epidermal keratinocytes by the oncogenic virus SV40 is a stage-specific process in which normal patterns of differentiation are progressively altered over time following infection. Within the context of this scheme, we examined the keratins produced by the infected cells. Immunofluorescence studies indicated that viral infection led to the formation of variant cells visibly lacking the normal keratin cytoskeleton after about 10-15 serial passages (60-90 cell generations) post infection. Analyses of variant cell formation in clonal populations grown on palladium islands revealed that the variants were derived within 2-3 cell divisions from cells containing an apparently normal keratin cytoskeleton, but that variant formation depended upon cell density. Immunoprecipitation of 35S-methionine labelled keratins from the infected keratinocytes revealed a gradual loss of the normal 46, 50, 56 and 58Kd keratin species over a period of many months after infection. The loss of the normal keratins was accompanied by the appearance of at least two species in the 48-52Kd size range not present in uninfected cells and the enhancement of a third, 40Kd, protein quite early after infection. Analysis of the altered keratin patterns on two-dimensional acrylamide gels using either isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHG) along the first dimension showed that the infected cells produced basic keratins which increased in relative abundance as cells became more transformed with serial passage including at least five isoelectric forms not seen in uninfected cells. Translation of poly A+ RNAs from the infected cells indicated that the altered keratin synthesis probably reflects changes in the translatable mRNA pool.     

Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector

Summary We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34°C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca⁺⁺-resistant cells did not proliferate at the nonpermissive temperature (40°C), indicating that they depended on T antigen for their proliferation. The temperaturesensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40°C. The serum/Ca⁺⁺-resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca⁺⁺-resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that ΔE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.