A novel human gene closely related to the abl proto-oncogene.

A DNA sequence related to the abl proto-oncogene was identified in human placenta. Molecular cloning and nucleotide sequence analysis revealed two putative exons whose predicted amino acid sequence was most homologous to the corresponding sequences of c-abl and v-abl but was related to other tyrosine kinase genes as well. The new sequence was localized by in situ hybridization and somatic cell genetic analysis to human chromosome 1q24-25, which differs from the location of any previously identified tyrosine kinase gene. The detection of a novel 12-kb transcript by this gene in human normal and tumor cells establishes it as a new member of the tyrosine kinase family that is closely related to but distinct from c-abl.

Correlation of morphological transformation to sister chromatid exchanges induced by split doses of chemical or physical carcinogens on cultured Syrian hamster cells.

The relationship between the induction of DNA damage as reflected by sister chromatid exchange (SCE) formation and morphological transformation in exponentially growing Syrian hamster embryo cells was determined quantitatively after split doses of chemical or physical carcinogens. With split doses of carcinogen separated by 2 to 24 hr, only N-acetoxy-2-fluorenyl-acetamide (0.50 microgram/ml) enhanced both SCE induction and transformation when compared to single exposure. Split doses of N-methyl-N'-nitro-N- nitrosoguanidine (0.20 microgram/ml), mitomycin C (50 ng/ml), or ultraviolet light (3.0 J/sq m) were less effective than single exposures, while split doses of methyl methanesulfonate (40 micrograms/ml) caused transformation frequencies similar to a single treatment and decreased SCE frequencies with time intervals greater than 4 hr. Split or single exposures of X-irradiation (200 R) resulted in similar low frequencies of transformation and SCE. Contrasting with these results, a significant potentiation of SCE occurred after split doses of N-methyl-N'-nitro-N-nitrosoguanidine in cultures arrested in G1 with arginine-glutamine-deficient medium or by contact inhibition compared to a single treatment. This response was attributed to the interaction of carcinogen with DNA containing unrepaired damage and demonstrates the importance of the cell cycle phase of the target cell during carcinogen exposure for the induction of SCE by split doses of N-methyl-N'-nitro-N-nitrosoguanidine. The similarity of responses for transformation and SCE induction with split doses of carcinogens suggests that DNA lesions involved in SCE are essential for the initiation of neoplastic development.

Chromosome alterations associated with in vitro exposure of human fibroblasts to chemical or physical carcinogens.

Normal human foreskin fibroblasts treated in vitro with a chemical carcinogen or irradiated with ultraviolet light subsequently acquired anchorage independent growth and an extended but finite capacity for exponential growth. All cell lines were derived from cells recovered from colonies that had grown in semisolid medium; cell lines originally treated with a chemical carcinogen produced nodules after s.c. inoculation into nude mice. G-banding analysis of 10 cell lines (including one ultraviolet light line) revealed that seven were chromosomally abnormal with structural and numerical chromosome alterations, one was characterized by a consistent trisomy, and the other two were normal diploid. Structural alterations consisted of chromosome deletions, translocations, and partial chromosome duplications. Although no common structural or numerical abnormality was detected, several structural alterations were observed involving chromosomes 1, 7, 11, and 22, where fgr, erb-B, H-ras-1, and sis protooncogenes, respectively, are located. In one cell line trisomy 17 was a unique chromosome alteration. The induction of chromosome changes may have influenced the proliferative capacity of the treated cells relative to nontreated cells. However, the two cell lines without detectable chromosome changes also had an increased proliferative life span, suggesting that other submicroscopic genetic alterations may have affected cell multiplication. Although carcinogen induced chromosome abnormalities may represent a step in the process of neoplastic development, additional genetic and/or epigenetic changes, are required for indefinite growth and the expression of malignancy.

Expression and chromosomal localization of the cytochrome P1-450 gene in human mitogen-stimulated lymphocytes.

Genetic differences in aryl hydrocarbon hydroxylase (AHH) (flavoprotein-linked monoxygenase EC 1.14.14.1) activity in cultured lymphocytes have been linked with individual risk for certain environmentally caused cancers. Cytochrome P1-450 is the form of cytochrome P-450 most closely associated with AHH activity. In this study the chromosomal localization and the expression of human cytochrome P1-450 gene were determined in phytohemagglutinin-stimulated lymphocytes. In situ hybridization analysis provides assignment of the structural gene for human cytochrome P1-450 to chromosome 15q22-q24. Treatment of lymphocytes with benzanthracene increased the amount of mRNA hybridized to the cloned cytochrome P1-450 gene. The level of cytochrome P1-450 mRNA in these lymphocytes correlates well with the induced AHH activity indicating that non-cytochrome P1-450 enzymes contribute little to the individual differences in the level of AHH activity in the lymphocytes. Southern analyses of genomic DNA from individuals with high and low induced AHH activity demonstrated no detectable differences in the pattern or intensity of restriction fragments after treatment with benzanthracene from either individual. This finding together with the excellent correlation between the induced cytochrome P1-450 and AHH activity, suggests that transcriptional control rather than gene amplification or gross form of gene rearrangement accounts for cytochrome P1-450 induction in man. Measurements of cytochrome P1-450 mRNA content in cultured lymphocytes provide an alternative approach to the assay of AHH activity in assessing AHH phenotype and predicting different susceptibilities to deleterious environmental agents.

Human papillomavirus type 18 DNA is integrated at a single chromosome site in cervical carcinoma cell line SW756.

SW756, a cervical carcinoma cell line, has multiple copies of human papillomavirus type 18 DNA sequences. The integration site of human papillomavirus type 18 DNA was localized by in situ hybridization to chromosome 12 at band q13. This single integration site corresponds to a heritable fragile site, which may have facilitated the integration of the viral DNA.

Integration sites of human papillomavirus 18 DNA sequences on HeLa cell chromosomes.

Human papillomaviruses (HPV) 16 and 18 are closely linked with human genital cancer. In most cervical carcinomas, viral sequences are integrated into the host genome. HeLa, a cervical carcinoma cell line, has multiple copies of integrated HPV 18 DNA. In this study, in situ chromosome hybridization was used to assign the integration sites of HPV 18 DNA sequences on HeLa cell chromosomes. Four sites of hybridization were identified at 8q23----q24, 9q31----q34, p11----p13 on an abnormal chromosome 5, and q12----q13 on an abnormal 22. Three of these sites correspond with the locations of MYC, ABL, and SIS protooncogenes, and are at or in close proximity to fragile sites. The chromosomal localization of HPV 18 DNA may be useful in assessing the role of viral integration in the development of this malignancy.

Antipain inhibits N-methyl-N'-nitro-N-nitrosoguanidine-induced transformation and increases chromosomal aberrations.

The morphologic transformation induced in Syrian hamster embryo cells by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0.25 microgram/ml of medium) is inhibited by posttreatment with antipain (6-600 microgram/ml), a protease inhibitor, but is unaffected by pretreatment. DNA replication relative to untreated controls is not affected by MNNG, antipain, or the combination of the two; no synergistic lethality of antipain and MNNG occurred as reflected in the cloning efficiency. Antipain was ineffective in influencing MNNG-induced sister chromatid exchanges, but it increased frequencies of chromosomal aberration (per metaphase) at 10, 26, and 40 hr when cells were treated with MNNG at 0.25 microgram/ml of medium followed by antipain 10 min later, the procedure used in the transformation studies. Antipain also increased the average number of aberrations at the second mitosis (34 hr) when the MNNG concentration was doubled. Chromatid exchanges increased 26 hr posttreatment with the combination of MNNG and antipain used for transformation. No difference in MNNG-induced aberrations was observed when antipain preceded MNNG by 24 hr. Although the mode of actin of antipain is unknown, antipain does not inhibit transformation by suppressing chromosomal rearrangements that could convert recessive mutations to the homozygous state.

Enhancement of N-methyl-N'-nitro-N-nitrosoguanidine transformation of Syrian hamster cells by a phorbol diester is independent of sister chromatid exchanges and chromosome aberrations.

12-O-Tetradecanoylphorbol 13-acetate (TPA), a known tumor promoter, enhances the morphologic transformation of Syrian hamster embryo cells induced by low transforming concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0.025-0.1 microgram/ml) without potentiation of cell lethality or of changes in sister chromatid exchanges (SCEs) or chromosomal aberration frequencies. When MNNG was added to logarithmically growing cultures and TPA was added 2, 24, or 48 hr later, no changes in SCE frequency relative to MNNG alone occurred. Similar results were obtained with TPA in cells that had been exposed to MNNG in stationary growth phase. Whereas no transformation occurred with TPA alone and pretreatment with TPA did not affect MNNG transformation, its addition 2 hr after MNNG reduced transformation frequency but addition 24-72 hr after MNNG increased the transformation frequency up to 6-fold. TPA had a minimal effect on increasing the transformation frequency (2-fold) induced by MNNG at 0.25 micrograms/ml, a high concentration. Of three polycyclic hydrocarbons, perylene, benzo[g,h,i]perylene, and benz[a]anthracene, known as weak or noncarcinogens, only benz[a]anthracene induced a very low transformation frequency; however, after TPA, transformation occurred with all three. Because the number of cells whose transformation was initiated by low doses of carcinogen is much larger than the number of cells giving rise to transformed colonies in the absence of TPA, the frequency of the initial event is greater than can be expected from point mutations. Furthermore, the promotional aspect of transformation is not accompanied by a parallel increase in SCE.

Human and rodent transformed cells are more sensitive to in vitro induction of SCE by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) than normal cells.

The sensitivity to sister chromatid exchange (SCE) induction by N-methyl-nitro-N'-nitrosoguanidine (MNNG) in human, mouse, Chinese hamster, and Syrian hamster normal cell strains and in permanent transformed cell lines of the same species was compared. Exponentially growing or growth-inhibited cultures of permanent cell lines transformed spontaneously or by chemical carcinogen or oncogenic virus responded with a higher SCE frequency after MNNG treatment than did normal diploid cell strains. Compared with the normals, exponentially growing Simian virus 40 transformed human fibroblast GM637 had the highest SCE frequency, followed by mouse cell line alpha L929 and Chinese hamster V79-4; the least sensitive were two Syrian hamster cell lines, OBP, derived from transformation of embryo cells with benzo(a)pyrene, and BHK, a spontaneously transformed baby hamster kidney line. Similar results were obtained with cultures arrested in G1 with glutamine-arginine deficient medium. The SCE response observed with transformed cells to carcinogen probably reflects cellular changes associated with the transformed state, such as shortening of the cell cycle, excision repair deficiency, or an increase in DNA replicon size. The current results demonstrating a difference in SCE induction between normal and malignant cells are important since normal or transformed cultured cells are utilized to assess potentially deleterious environmental agents, particularly carcinogens. In general, SCE induction by a specific direct acting carcinogen may be a useful approach for identifying transformed cells with the ability to produce tumors.

Relationship of carcinogen-induced sister chromatid exchange and neoplastic cell transformation.

Sister chromatid exchange (SCE) and morphological transformation induced by five chemical carcinogens, N-acetoxy-2-fluorenyl-acetamide (AcAAF), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), benzo[a]-pyrene (BP), and cisplatinum(II) diamine dichloride (PT) as well as by X-irradiation were quantified in parallel experiments with cultures of Syrian hamster embryo cells (HEC). Incubation of HEC with 5-bromodeoxyuridine (10(-5)M) for two rounds of replication (24 h) required for SCE visualization neither caused morphological transformation nor altered the transformation frequency induced by carcinogen alone. All chemical carcinogens, but not X-irradiation, produced a dose-dependent increase in SCE and transformation frequency, demonstrating the sensitivity of both assays to carcinogens. The ratios of induced SCE relative to transformation frequency, however, varied with the carcinogen. BP, MMNG, and AcAAF were similarly efficient in inducing SCE compared to transformation but were considerably less effective than MMS and PT. X-irradiation at doses of 200, 300, and 500 R did not cause transformation and induced a low frequency of SCE. On a molar basis, MMS and PT were the most effective in SCE induction relative to transformation, MNNG and AcAAF were less effective, and BP was the least effective carcinogen. The positive linear correlation between chemical carcinogen-induced SCE and transformation suggests a relationship between the two cellular responses.

Persistence of sister chromatid exchanges and in vitro morphological transformation of Syrian hamster fetal cells by chemical and physical carcinogens.

The induction of neoplastic cell transformation is closely associated with DNA alterations which occur shortly after carcinogen exposure. Sister chromatid exchange (SCE) formation is a sensitive indicator of carcinogen-DNA interaction and correlates with the induction of morphological cell transformation. The persistence of lesions generating SCE produced by chemical and physical carcinogens and its relevance to the induction of morphologic transformation was evaluated in coordinated experiments with cultured Syrian hamster fetal cells (HFC). Exponentially growing HFC were exposed for 1 h to benzo[a]pyrene (BP), methyl-methanesulfonate (MMS), cis-platinum (II) diaminedichloride (cis Pt II), N-methyl-N'-nitrosourea (MNU), mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-acetoxy-2-fluorenyl-acetamide (AcAAF) or u.v. light irradiated. Cells were incubated for 24 h with 5-bromodeoxyuridine (BrdUrd) required for SCE visualization at 1, 24 and 48 h after carcinogen exposure. The induction of morphological transformation was determined on the quantitative colony assay at 6 days after carcinogen treatment. SCE analysis demonstrates that for a period of 48 h after carcinogen exposure, during which time the cells undergo at least four replicative cycles, DNA damage generating SCE induced by all chemical carcinogens either persisted or was partially removed, whereas u.v.-induced lesions were completely removed. An elevated SCE frequency persisted after two additional cell cycles after treatment with BP, AcAAF or MMC without increased cell lethality as compared to other carcinogens whose lesions were completely eliminated during the same period. Although a correlation between the persistence of SCE and the induction of transformation was not observed for all carcinogens, this study illustrates that DNA damage generating SCE can persist over several replicative cycles, thus raising the possibility that lasting DNA alterations are important for the induction of neoplastic cell transformation.

Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms.

Amplification of the erbB/EGF receptor and a structurally related gene, designated erbB-2, have previously been detected in a variety of human tumors. In a series of human mammary tumor cell lines, analysis of transcripts of these genes revealed elevated levels of one or the other in more than 60% of tumors analyzed. Eight cell lines demonstrated erbB-2 mRNA levels ranging from 4- to 128-fold above those of normal controls. erbB-2 expression was evaluated in comparison to the expression level of actin observed in these cell lines. There was no evidence of an aberrantly sized erbB-2 transcript in any of these lines. Immunoblot analysis indicated elevation in levels of the 185-kd product of the erbB-2 gene expressed by these cells. In four lines erbB-2 gene amplification in the absence of an apparent gene rearrangement was demonstrated. In a representative cell line of this type, SK-BR-3, the amplified erbB-2 gene copies were located in an aberrant chromosomal location. Four additional cell lines, which demonstrated 4- to 8-fold overexpression of erbB-2 mRNA, did not exhibit gene amplification. In a representative cell line of this type ZR-75-1, an apparently normal chromosomal location was found for the erbB-2 gene. Our findings indicate that overexpression of the erbB-2 gene in mammary tumor cell lines is frequent and associated with different genetic abnormalities.

12-O-tetradecanoyl-phorbol-13-acetate and its relationship to SCE induction in Syrian and Chinese hamster cells.

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) in conditions that produce enhancement of ultraviolet light (UV) and x-irradiation Syrian hamster embryo cell (HEC) transformation did not cause further increase in the sister chromatid exchange (SCE) frequency induced by UV and x-irradiation, two physical carcinogens that differ in their mode of DNA interaction and efficiency of SCE induction. Several factors which might influence SCE induction by TPA were studied on HEC and Chinese hamster V79-4 cells. Heat-inactivated serum was used because of the possibility that a serum component may interfere with TPA ability to cause SCE. TPA effect on SCE was determined at the first and second division post treatment on cells exposed to different 5-bromodeoxyuridine (BrdUrd) concentrations. Independent of BrdUrd concentration (1-10 microgram/ml medium) and the number of cell divisions post treatment TPA (0.01-2 microgram/ml medium) was ineffective in inducing SCE in exponentially and stationary HEC cultures cultivated in medium supplemented with heat-inactivated serum. Also, TPA did not increase the SCE frequency in V79-4 Chinese hamster cells cultured in heat-inactivated or noninactivated serum. Although SCE induction, a cellular response to carcinogen-induced DNA damage, may be important for the induction of transformation by environmental agents, the enhancement of transformation frequency caused by TPA occurs without further DNA alterations involved in SCE formation.

Induction of chromosome banding by trypsin/EDTA for gene mapping by in situ hybridization.

We describe an easy and reproducible procedure that utilizes trypsin/EDTA for the induction of chromosome banding in conjunction with in situ hybridization. The high quality banding resolution required for grain localization is obtained on both elongated and contracted chromosomes derived from synchronized or nonsynchronized human lymphocytes or fibroblasts. This procedure can also be useful for gene localization on chromosomes from cancer cells.

Chromosomal localization of three human ras genes by in situ molecular hybridization.

Three human ras family protooncogenes, c-Ki-ras-1, and c-Ki-ras-2, and N-ras, have been mapped to chromosome bands 6p11-12, 12p11.1-12.1, and 1p11-13, respectively by in situ molecular hybridization. Certain human cancers display consistent and specific alterations involving chromosomes 1, 6, and 12. The precise chromosomal localization of ras genes will permit evaluation of the possible effect of these chromosome changes on the structure and activities of ras protooncogenes in human neoplasia.

Chromosome distribution of intracisternal A-particle sequences in the Syrian hamster and mouse.

Metaphase chromosomes of Syrian hamster and BALB/c mice were hybridized in situ with radiolabeled probes derived from cloned intracisternal A-particle (IAP) genes of the corresponding species. The DNAs of these species are known to contain about 900 and 1,000 copies, respectively, of the retrovirus-like IAP sequence elements per haploid genome. Multiple IAP sequences were found on all chromosomes of both hamster and mouse. In the hamster, more than half of the IAP sequences were located in regions of non-centromeric constitutive heterochromatin, at an average concentration per unit chromosome length 5 times greater than in the euchromatic regions. The other dispersed sequences showed marked local variations in concentration along the chromosome lengths; both discrete foci and large grain clusters were observed as well as regions apparently lacking IAP sequences. Within the resolution of the techniques, IAP sequences appeared to be more evenly distributed over the mouse chromosomes; however, some prominent variations in concentration were seen. The number of potentially active IAP genes in the Syrian hamster, and by extension in the mouse, may be restricted by the preferential location of IAP sequences in genetically inert regions of the genome.

Isolation and chromosomal localization of the human fgr protooncogene, a distinct member of the tyrosine kinase gene family.

The cell-derived domain of Gardner-Rasheed feline sarcoma virus (GR-FeSV) consists of a gamma-actin- and a tyrosine-specific protein kinase-encoding sequence designated v-fgr. By utilizing a v-fgr probe, it was possible to detect related sequences present at low copy number in DNAs of a variety of mammalian species and to isolate a human fgr homologue. Comparative studies revealed that this human DNA clone represented all but 200 base pairs of v-fgr. Analysis of human genomic DNA demonstrated that the fgr protooncogene was distinct from the cellular homologues of other retrovirus onc genes. In addition, the fgr protooncogene was localized to the distal portion of the short arm of human chromosome 1 at p36.1-36.2 by in situ hybridization. Taken together, our findings establish that the fgr protooncogene is a unique member of the tyrosine kinase gene family.