Vittorio Defendi

Simian virus 40-induced chromosome changes in human epidermal cultures

Abstract Chromosomal aberrations in human epidermal keratinocytes are described for the first time; the cells were transformed in vitro by the oncogenic virus, SV40 (Simian virus 40). The majority of metaphases exhibited hypotetraploid chromosome numbers and configurations indicative of frequent chromosomal breakage and fusion(s). The abnormalities were associated with segregational errors during various stages of mitosis; these errors were about 30 times as frequent in the transformed cells as in the uninfected keratinocytes. The pattern of karyotypic changes induced by the SV40 virus is similar to that previously observed in infected human fibroblasts, indicating that the alterations are viral-driven rather than cel-determined.

Amplification and rearrangement of integrated SV40 DNA sequences accompany the selection of anchorage-independent transformed mouse cells

Clones of SV40 tsA mutant-transformed mouse embryo cells with a temperature-independent transformed growth phenotype were derived from a parental line with a temperature-dependent growth phenotype, using the selective pressure of anchorage independent growth in agar at 40 degrees C. The parental J78 clone contained a complex tandem structure of integrated SV40 DNA within two cellular DNA fragments of 10 and 14 kb, generated by Bgl II digestion. The new clonal derivatives, in addition to an altered growth phenotype, displayed additional high molecular weight sites of SV40 DNA integration. A marked structural similarity among integration sites suggested that the new sites may have arisen by duplication and translocation of an original integration structure, or via an unequal crossover event. The rearrangement of viral DNA sequences appears to be specifically associated with the emergence of temperature-independent clones, since the isolation of new clones under nonselective conditions, that is, growth in semi-solid medium at 33 degrees C, was not accompanied by modification in cellular sequences containing SV40 DNA.

Re-expression of differentiated properties in SV40-lnfected 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.

Necessary and sufficient conditions for recruitment of macrophages into the cell cycle.

Abstract Mouse peritoneal macrophages whether harvested from a stimulated or unstimulated peritoneal cavity are mitotically quiescent. Either of these populations can be subdivided based upon the particular conditions needed to bring particular subsets back into the cell cycle. Three subsets of macrophages are evident in exudates obtained after stimulation in vivo with starch, endotoxin, or thioglycolate medium. About 50% of such cells proliferate in vitro in response to macrophage growth factor (MGF). For about one-third of MGF-responsive cells, a non-specific agent given in vitro is sufficient for recruitment into the cell cycle (subset 1). Non-specific agents given were dextrans, endotoxin, or thioglycolate. For the remaining two-thirds of MGF-responsive cells, it appears that a need for MGF must be fulfilled in vitro (subset 2). There is always a fraction of macrophages from stimulated peritonea that appear to be non-responsive (subset 3). Unstimulated animals yield two subsets of macrophages in culture: about 80–90% non-responsive cells (subset a) with the remaining fraction only responsive to MGF and a non-specific agent given in combination (subset b). It is possible that growth responsiveness may be used as a criterion to define macrophage activation at the single-cell level within populations that on the whole are activated or normal.

The differential effect of interferon on T antigen production in simian virus 40-infected or transformed cells

Abstract The ability of interferon (IF) to inhibit T antigen (Ag) production in simian virus 40 (SV40)-infected or transformed cells was studied primarily through the use of immunoprecipitation followed by gel electrophoresis and autoradiography. Addition of IF to monkey cells prior to or subsequent to inoculation with SV40 resulted in an inhibition in the amount of T Ag that was synthesized late in infection. In contrast when a similar experiment was performed with a is mutant of SV40, tsA58, which does not replicate at the nonpermissive temperature, there was no inhibition in the amount of immunoprecipitable T Ag when IF was added at 30 hr postinfection at 40.5°. The effect of IF on an integrated versus nonintegrated genome within the same cell population was studied in an SV40-transformed mouse cell, H6-15, which is temperature sensitive for the transformed phenotype and for expression of T antigen. In shift-down experiments it was shown that the reappearance of SV40 T Ag was insensitive to the addition of IF whereas superinfection of these same cells with polyoma virus resulted in a dose-dependent inhibition of polyoma T Ag infection. An SV40-transformed mouse cell line (nonpermissive) and two SV40-transformed human cell lines (semipermissive) were passaged in the presence of IF for four generations. Approximately the same amount of labeled T Ag could be immunoprecipitated from IF-treated compared to control mouse cultures whereas, there was a marked decrease in the amount of newly synthesized T Ag in IF-treated human cultures. All these results are compatible with the hypothesis that IF affects differentially the expression of early viral genes whether the viral DNA is integrated or not integrated.

Fusion-induced differentiation of SV40-infected human keratinocytes☆

Abstract 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.

The effect of interferon on the early functions of simian virus 40

Abstract The effect of interferon (IF) on the early functions of simian virus 40 (SV40) was studied by monitoring the inhibition of SV40-induced T antigen (Ag) in permissive monkey and nonpermissive mouse cells. When IF was added to cells which had previously been transformed by SV40, there was no effect on T Ag production. However, pretreatment of the cells with homologous IF 20 hr prior to inoculation with SV40 resulted in a dose-dependent decrease in the proportion of T antigen-positive cells at 48 hr postinfection. The kinetics of inhibition by IF of SV40-induced T Ag was studied in both cell types. When IF was added at increasingly later times after SV40 inoculation, there was a progressive reduction of T Ag-positive cells. Penetration and uncoating of SV40 were ruled out as the major site of IF action since the induction of T Ag by infectious DNA extracted from SV40 virions was also sensitive to treatment with IF. In addition, although IF may be added to the cells at a particular time, it is apparent that these cells do not become resistant to viral infection until several hours later. The results suggest that the inhibitory effect of IF is primarily at a step subsequent to the uncoating of SV40.

Expression of the E2 open reading frame of papilloma viruses BPV1 and HPV6b in Escherichia coli

A new expression vector, pRA10, has been constructed for the expression of the open reading frames (ORF) of bovine (B) and human (H) papilloma viruses (PV). This vector is a derivative of pAJ pi and contains 15 restriction sites proximal to the lambda PL promoter, offering considerable versatility for insertion of different ORFs. This vector was used specifically to express the E2 ORF gene products from BPV1 and HPV6b at high level in Escherichia coli. The genuine nature of these proteins was demonstrated by restriction map analysis of expression vector plasmids to insure proper orientation, nucleotide sequence analysis to demonstrate in-frame insertion, E2 ORF protein production by expression-vector plasmids, and not by appropriate controls and, immunoprecipitation of E2 proteins by antibody specific for a common N-terminal sequence derived from the expression vector.

The effects of serum concentration on the level of integration of simian virus (SV40) genome and the transformation frequency in SV40-infected Chinese hamster cells.

Abstract Treatment of Chinese hamster embryo cultures with different serum concentrations during the first 48 hr after simian virus 40 (SV40) infection, modified the proportion of cells in DNA synthesis, the amount of SV40-DNA integrated, the proportion of cellular DNA with 5-bromodeoxyuridine substitution in both strands, the proportion of polyploid cells, and the transformation frequency. All these parameters increase in a coordinated way as function of the serum concentration. It is suggested that the increase in the number of cells recruited into DNA synthesis facilitates the integration of viral DNA with an increased probability of transformation.

Transformation by human papillomavirus type 16 (HPV16) DNA but not HPV6b DNA is enhanced by addition of the human cytomegalovirus enhancer

Primary human cervical epithelial cells immortalized by human papillomavirus type 16 (HPV16) DNA exhibit altered morphology and differentiation characteristic of transformation, but show a lack of transformed phenotype relative to HPV18 DNA immortalized cells in terms of anchorage-independent growth (Pecoraro, Lee, Morgan, and Defendi, 1991, Am. J. Pathol. 138, 1-8). This is completely corrected by inserting a strong heterologous enhancer derived from human cytomegalovirus DNA upstream from the HPV16 long control region. The cells immortalized by this DNA form colonies in agar comparable to those formed by HPV18 DNA immortalized cells. The enhanced transformation capability correlates with increased levels of HPV16 E6-E7 and E5 transcripts. The HPV16 DNA containing this strong enhancer also transforms C127 mouse cells with increased efficiency and strength relative to the natural HPV16 DNA, as measured by the numbers and size of the colonies in agar. The positive effects of this strong enhancer appear specific for HPVs associated with genital malignancies such as HPV16, since HPV6b DNA (primarily in benign tumors) with or without the strong cytomegalovirus enhancer is incapable of immortalizing primary human cervical epithelial cells or allowing efficient growth of C127 mouse cells in agar. These results suggest that the diminished oncogenic properties of HPV16 versus HPV18 DNA in cultured cells and in human malignancies may reside in the long control regions of these viruses and, additionally, may define another difference in the oncogenic properties of HPVs associated with benign or malignant genital neoplasia.