Joseph A. DiPaolo

Preferential sites for viral integration on mammalian genome

Chromosomal localization of human papillomavirus (HPV) 16 and 18 on human cervical carcinomas and epithelial cell lines obtained after HPV transfection has uncovered a nonrandom association of viral integration and specific genome sites. Fragile sites appear to be preferential targets for viral integration because of their structural and functional characteristics through which chromosomal anomalies, alterations in protooncogene activity, and gene amplification can occur. Individually or in association, such changes lead to the acquisition of an unlimited cell growth potential but not tumorigenicity. Genetic instability and uncontrolled cell division resulting from HPV integration increase the cells susceptibility to other exogenous carcinogenic factors that may complete the process of neoplastic development.

Viral integration, fragile sites, and proto-oncogenes in human neoplasia

SummaryTo evaluate the trend of viral integration in the human genome, chromosomal localization of five DNA-containing viruses compiled from literature data was compared to the location of fragile sites and protooncogenes. A total of 35 regionally mapped viral integration sites from tumors and transformed cells were distributed over 19 chromosomes. Of the 35 integration sites 23 (66%) were at the bands of fragile sites, and 7 were one band away (20%). This statistically defines the correlation as highly significant (P = 0.0000183, Fishers F-test). Five integration sites did not correspond to the location of a fragile site. Thirteen integration sites and proto-oncogenes mapped at the same bands (37%), 6. (17%) were one band apart, and at 16 integration sites (46%) no proto-oncogenes were localized (P = 0.00491). Eighteen viral integration sites, fragile sites, and protooncogenes (51%) were localized at the same bands or one band distant. This clustering of viral integration sites, fragile sites, and proto-oncogenes is statistically highly significant (P = 0.0000118), and indicates nonrandom viral integration in the human genome.

Cis-platinum(II) diamine dichloride causes mutation, transformation, and sister-chromatid exchanges in cultured mammalian cells.

The anti-tumor agent cis-platinum(II) diamine dichloride caused dose-dependent toxicity in V79 Chinese hamster cells and in secondary Syrian hamster embryo cells. Chromosome aberrations were induced and positive dose--response relationships were observed for induction of sister-chromatid exchanges and 6-thioguanine-resistant mutations in V79 cells and morphologic transformation of secondary Syrian hamster embryo cells. The findings suggest that this chemical is a potential human carcinogen.

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.

Refined localization of the erbB-3 proto-oncogene by direct visualization of FISH signals on LUT-inverted and contrast-enhanced digital images of DAPI-banded chromosomes

Contrast-enhanced, look-up-table (LUT)-inverted digital images of DAPI-banded chromosomes after fluorescence in situ hybridization (FISH) permit direct regional chromosomal localization of the fluorescent signals of single-copy gene probes. Improved quality and resolution of chromosome banding allowed a refined localization of the erbB-3 protooncogene from chromosome band 12q13 to sub-bands 12q13.2-13.3. This procedure can be used for direct and precise mapping of single-copy genes on both normal and cancer-cell-rearranged chromosomes.

HSV-2-induced tumorigenicity in HPV16-immortalized human genital keratinocytes

A subgenomic region of HSV-2, BglII N, is capable of converting immortal genital epithelial cells containing integrated HPV16 sequences into tumorigenic squamous cell carcinoma cells. Moreover, tumor-derived cultured cells and immortal cells that had been transfected with HSV-2/Bg/II N and kept in continuous culture subsequently lost the HSV-2 sequences. The HSV-2/Bg/II N sequence was ineffective on normal cells. Thus, HSV-2/Bg/II N may act as a cofactor in the genesis of a carcinoma but is not required to maintain the transformed phenotype. Although papillomaviruses (HPVs) are currently receiving much attention because of their association with cervical squamous carcinomas, ample reasons exist to suggest a multifactorial etiology in which additional factors are necessary to convert dysplastic lesions to carcinomas. The hypothesis that specific HPV types may be necessary but not sufficient to cause cancer is reinforced. Thus, HSV-2-transforming sequences have a potential role in the etiology of human cervical cancer.

Sarcoma-Producing Cell Lines Derived from Clones Transformed in vitro by Benzo[a]pyrene

Cells derived from mixed Syrian hamster embryo cultures were treated with pyrene (control) or with benzo[a]pyrene. Transformed clones were obtained only with the carcinogen. Some of the transformed clones were responsible for cell lines that produced tumors when injected into hamsters. These observations provide evidence that chemical-induced oncogenesis can be studied by an in vitro model.

POLYDACTYLISM IN THE OFFSPRING OF MICE INJECTED WITH 5-BROMODEOXYURIDINE.

The intraperitoneal injection of multiple doses of 5-bromodeoxyuridine (BUDR) into pregnant mice produced abnormalities limited to the hind limbs of the offspring. This effect is probably a result of a metabolic lesion. When injected into newborn mice, BUDR did not increase the incidence of tumors.

Two type II keratin genes are localized on human chromosome 12

SummaryHuman genomic DNA containing two type II keratin genes, one coding for keratin 1 (K1, a 68-kD basic protein) and another closely linked type II gene 10–15 kb upstream (K?, gene product unknown), was isolated on a single cosmid clone. EcoRI restriction fragments of the cosmid were subcloned into pGEM-3Z, and specific probes comprising the C-terminal coding and 3′ noncoding regions of the two genes were constructed. The type II keratin genes were localized by in situ hybridization of the subcloned probes to normal human lymphocyte chromosomes. In a total of 70 chromosome spreads hybridized with the K? probe (gHK?-3′, PstI, 800 bp), 36 of the 105 grains observed were on chromosome 12, and 32 of these were clustered on the long arm near the centromere (12q11–13). In 100 labeled metaphases hybridized with the K1 probe (gHK1–3′, BamHI-PstI, 2100 bp), 53 grains localized to chromosome 12 and 46 of these were found in the same region (q11–13). Therefore, both the gene for human keratin 1, a specific marker for terminal differentiation in mammalian epidermis, and another closely linked unknown type II keratin gene (K?, 10–15 kb upstream of K1) are on the long arm (q11–13) of human chromosome 12.

Asbestos and Benzo(a)pyrene Synergism in the Transformation of Syrian Hamster Embryo Cells

Four varieties of asbestos fibers, crocidolite, anthophyllite, amosite, and chrysotile, induced a low rate of morphologic transformation in Syrian hamster cells. Of the four tested, chrysotile was the most lethal as reflected by colony survival. When cells were exposed to 1 microgram of benzo(a)pyrene (BP)/1 ml medium or 3 J/m2 ultraviolet irradiation and to different concentrations of the asbestos fibers, an enhancement of transformation occurred only with BP. The enhancement was dose responsive with all fiber species except for amosite which was dose independent. The synergistic activity of BP and asbestos suggests that asbestos facilitates the transport of BP to the cell site(s) critical for transformation. These results provide a basis for investigating the carcinogenic and cocarcinogenic potential of asbestos fibers in mammalian cells.