Restriction mapping and the determination of scattered nucleotide sequences have permitted a description of the global structure and evolutionary affinities of the canine herpesvirus (CHV) genome. The global structure closely resembles that of the totally sequenced genomes of varicella-zoster virus and equine herpesvirus 1 (EHV-1) in having a 37 bp inverted repeat flanking a long unique region (UL) of approximately 100,000 bp, and a 10,100-10,700 bp inverted repeat flanking a short unique region (U8) of roughly 7,400-8,600 bp. On the basis of the sequences obtained, 35 homologues to previously identified herpesvirus gene products were found in UL and the major inverted repeat, and the level of the similarities indicated that CHV belongs to the genus Varicellovirus. Within the genus, CHV appears to be most closely related to EHV-1, pseudorabies virus and feline herpesvirus. Surprisingly, genes for both subunits of the viral ribonucleotide reductase were found to be missing from their equivalent place in other herpesvirus genomes. Either they have been translocated to another position in the CHV genome or, we think more likely, they have been lost.

Eight LF × ICIG cell hybrid clones, isolated upon fusion of normal ICIG‐7 human fibroblasts with tumorigenic, non‐metastatic LF CI.2A cells derived from a DAB‐induced rat hepatocarcinoma, were studied. 'They were all highly tumorigenic and were capable of developing spontaneous lung metastases in syngeneic animals. All the hybrids were characterized by a rapid loss of human chromosomes. However, in long‐term culture, they all revealed a persistence of human genetic information as assessed by Southern blotting. In hybrid lines in which human chromosomes were still visible, the most recurrent were numbers 7 and 9. Neither chromosome 7, previously reported to bear some of the genes controlling metastasis in human × mouse T‐cell hybrids, nor chromosome 9 appeared to be correlated with the metastatic potential of LF × ICIG hybrids. The same conclusion applied (I) to a human 3.3‐kb EcoRI DNA fragment which was amplified (approx. 10‐fold) only in metastases induced by one out of 3 metastatic hybrids tested; (2) to the transcription level of c‐Ha‐ras and c‐Ki‐ras genes which was enhanced (approx. 4‐fold) in metastatic and non‐metastatic lines as well. Co‐transfection of LF CI.2A cells with pHSG 272 selectable marker DNA and genomic DNA from normal ICIG‐7 human cells or from a hybrid‐induced metastasis, reproducibly gave rise to geneticin‐resistant transfectants capable of producing spontaneous lung metastases. Neither transfectants nortransfectant‐induced metastases harbored detectable human DNA sequences but all harbored pHSG 272 DNA. These results again call for caution in gene transfer studies of the metastatic process.

Summary— Methionine‐dependent (Met − Hcy + ) revertant sublines were isolated from a methionine‐independent (Met + Hcy − ) rat hepatocarcinoma cell line (LF). Characterization of these sublines has shown that long‐term culture in methionine‐deprived medium significantly decreases their ability to grow in vitro , their tumorigenicity in new‐born syngeneic animals and their level of transcription of 3 oncogenes (c‐Ki‐ ras , c‐Ha‐ ras , c‐ myc ) involved in hepatic growth. These data strongly support a relationship between methioninedependence and ability to grow in vivo as tumors. A shift in methionine‐supplemented medium for several cell generations of the various sublines grown in methionine deprived medium, has revealed that under these conditions, the cells maintain a diminished in vitro growth and tumorigenic capacity, despite an increased transcription of c‐Ki‐ ras , c‐Ha‐ ras and c‐ myc . This points out the complexity of the relationship between the level of activity of some oncogenes and the expression of the malignant phenotypes.

The recessive autosomal hereditary disease, xeroderma pigmentosum (XP), is characterized by a high incidence of tumors in sun-exposed skin. The defect in early steps of excision repair of XP cells leads to hypermutability towards UV-mimicking agents. DNA from eight XP tumors were screened for activated transforming genes using 3T3 transfection. In two skin tumors isolated from a XP child, an activated N-ras oncogene was detected. Synthetic oligonucleotide probes were used to characterize the mutation in the ras gene. Both tumors were found to be mutated in the 61st N-ras codon from gln to his. The mutation was accompanied by an increase in the level of N-ras specific mRNA and in one transformant, by the alteration of the p21 protein. In the same tumors, c-myc amplification and over transcription, and Ha-ras gene rearrangement and amplification were also detected. Analysis of other XP tumors with eleven different oncogene probes revealed an amplification of the Ha-ras gene in 6 out of 10 cases. The normal skin fibroblasts from XP patients show normal pattern levels of N-ras, c-myc and Ha-ras sequences. The hypothesis is proposed that the presence of several oncogene alterations in the same tumor could be due to the high amount of UV-induced DNA lesions found in the exposed skin cells, in the absence of efficient repair.

The human HBL‐100 cell line harbours SV40 DNA integrated in tandem at a unique site. The SV40 T‐antigen expressed in these cells is defective in a function essential to the replication of the viral genome. The integrated SV40 sequences were molecularly cloned in a bacteriophage, and a subclone (plasmid pSVHBI) containing a complete SV40 DNA was isolated. As compared to SV40 wild‐type strain 776, sequence analysis of pSVHBI early region revealed the presence of several DNA alterations. Among these, a point mutation at position 3199, predicting a change at amino‐acid 540 of arginine to isoleucine, was shown by marker rescue to be responsible for the deficiency of T‐antigen. This novel mutation further delimits one of the T‐antigen domains involved in SV40 DNA replication. Transfection experiments demonstrated that the transforming activity of the SV40 genome from HBL‐100 cells is still preserved. Moreover, several transformed human cell clones thus obtained could be permanently established in culture.

The human osteosarcoma cell line Te85 clone F-5 is not tumorigenic in vivo. Its transformation with Kirsten murine sarcoma virus (KiMSV) (KHOS) confers full malignant properties and stable non-tumorigenic revertants of this KHOS cell line have been obtained. Here we show that integration and expression of a single copy of the KiMSV proviral DNA, which is totally lost in the HOS 240S revertant, is responsible for the acquisition of tumorigenicity. Cytogenetic analysis and the absence of a residual LTR copy in the revertant cellular genome suggest that the loss of KiMSV provirus is caused either by chromosomal segregation or by recombination not involving the LTR. In addition analysis of the expression of ras proteins revealed no changes in the pattern of c-ras products and the expression of v-ras only in the KHOS cells. All these data suggest that Te85 and HOS 240S cell lines could represent a human alternative recipient system to rodent cells in studies with oncogenes.

Cytogenetic analysis of the human HBL-100 cell line, that we have previously shown to harbour SV40 genetic information (Caron de Fromentel et al., 1985), reveals numerous chromosomal rearrangements as soon as the 30th in vitro passage. The karyotype is relatively stable during in vitro maintenance and even at late passages (approximately 70) when the cells have acquired the capacity to form tumors in nude mice. In all the somatic cell hybrids obtained after fusion of mouse 3T3-4E cells with HBL-100 cells, several human chromosomes are maintained and a derivative from chromosome 15-der(15)- is the most frequently observed. The der(15) marker is present in the HBL-100 cell line at every passage studied as well as in different cell lines derived from tumors induced by HBL-100 cells. The various hybrids, originally isolated for a transformed phenotype on the basis of their ability to grow in soft-agar, were all found to express the SV40 T-antigen. In situ hybridization of an SV40 DNA probe to chromosome spreads obtained from one of these hybrids shows that the integration site of the viral genome is located on the der(15) marker chromosome, at band 15q24. The possible cooperation of SV40 T-antigen with some other oncogene(s), required by human HBL-100 cells in order to express a malignant phenotype, is discussed.

The human epithelial HBL-100 cell line harbors SV40 genetic information and has an unlimited growth potential. Despite displaying properties characteristic of transformation since its early in vitro passages, it is capable of producing progressively growing tumors in nude mice only after long-term culture. This is a reproducible phenomenon and apparently not the consequence of a selection of preexisting malignant cells. Superinfection of early passage nontumorigenic HBL-100 cells with Kirsten murine sarcoma virus, which contains a Ki-ras oncogene having undergone multiple activating events, induces morphologic alterations and rapidly converts the cells to neoplastic cells, further supporting the hypothesis of multistep carcinogenesis. The HBL-100 cell line might be useful in defining the oncogenes representative of different families, which are able to complement SV40 in this system.

Three human tumor cell lines derived from an osteosarcoma (OHA cells), a bladder carcinoma (EJ cells), and a gastric sarcoma (SHAC cells) were passaged serially in the presence of human interferon-alpha (IFN-alpha) for extended periods of time. The long-term IFN-alpha treatment induced a partial reversion of OHA tumor cell phenotype as exemplified by inhibition of cell proliferation, lack of cellular overlapping in confluent cultures and marked reduction in tumorigenicity. In contrast, under the same conditions, long-term IFN treatment did not reverse but even potentiated some of the phenotypic characteristics (including tumorigenicity) of EJ and SHAC cells. In the three tumor cell lines, the transforming ability, genomic level, or expression of activated oncogenes, c-Ki-ras, c-Ha-ras, and N-ras, respectively, were unaltered with long-term IFN-alpha treatment. Our data indicate that IFN-induced phenotypic changes are not necessarily associated with changes in oncogene expression.

The prolongation of the limited lifespan of human embryonic lung fibroblasts by hydrocortisone was previously associated with increased uridine incorporation into ribosomal RNA (rRNA). Using polyacrylamide gel electrophoresis, we show here that the continuous treatment with 12 μM hydrocortisone results in increased uridine incorporation into rRNA precursors in cells stimulated to grow by serum, but not in resting cells, suggesting that the hormone does not directly activate rRNA transcription. Chase experiments indicate that the stability of 28S and 18S rRNA is enhanced by hydrocortisone in quiescent cells but not after serum refeeding. Similar results were also observed for transfer RNA (tRNA). Therefore, these effects of hydrocortisone seem to be indirect consequences of pleiotropic actions of glucocorticoid, such as stimulation of processes involved in cell proliferation and inhibition of lysosomal enzymes.

Antisera were raised against HeLa cells and mouse cells transformed after exposure to HeLa chromosomes (ME-ch.HeLa). The antisera were positive in indirect immunofluorescence assays on both HeLa and ME-ch.HeLa cells, but were negative on normal mouse cells. Immunoprecipitation of 125I-labelled cell extracts showed that ME-ch.HeLa cells contain at their surface 3 proteins of apparent molecular weights of 185,000, 105,000 and 45,000 daltons, which were also present on the surface of our HeLa cells but not on other mouse cell lines tested. However, further study has shown that these proteins are not normal constituents of HeLa plasma membranes but are in fact surface proteins of Mycoplasma orale.

Mouse embryo cells, transformed in vitro by the transfer of chromosomes from HeLa human tumour cells, express a surface antigen (s) also found on HeLa cells. This antigen(s), which has been detected both by indirect immunofluoresence and by a 125I-protein A binding assay, is not an antigen(s) shared by both Human and Mouse cells.

Rat epithelioid lung cells were transformed with various preparations of SV40 DNA using the Ca2+-precipitation technique. The amount of SV40 genetic information integrated into transformed clones was evaluated by DNA-DNA renaturation kinetics. The growth properties on plastic and in soft-agar were examined, as well as the ability to induce tumors in syngeneic new-born animals or in adult nude mice. One particular transformed line, which had received the HpaII/BamHI A (59 per cent) fragment, was found to contain about 3 integrated copies of this fragment per cell and no significant amount of the HpaII/BamHI B (41 per cent) fragment. This line which grew to high saturation densities and efficiently formed clones in low serum on plastic, produced tumors in both syngeneic rats and nude mice. Thus the HpaII/BamHI A fragment, which mainly includes early viral information, was sufficient to impart these properties to rat epithelioid lung cells.