Specific chromosome defect associated with human small-cell lung cancer; deletion 3p(14-23).

A specific, acquired chromosomal abnormality (deletion 3p) has been found in at least one chromosome 3 in 100 percent of the metaphases in 12 of 12 cell lines cultured from human small-cell lung cancer tissue and in 2-day tumor culture specimens from three patients. Analysis of the shortest region of overlap shows the deletion to be 3p(14-23). This specific change was not seen in five of five lung cancer cell lines other than small-cell lung cancer or in two lymphoblastoid lines cultured from cells of small-cell lung cancer patients whose tumors had the 3p deletion.

Cytogenetic studies of human breast cancer lines: MCF-7 and derived variant sublines.

Cytogenetic studies of the human breast cancer cell line MCF-7 and the sublines derived from it demonstrated extensive aneuploidy with both numerical and structural abnormalities and a wide range of heteroploidy. Detailed G-banding analyses showed that loss of marker chromosomes was a rare event, while formation of additional structural abnormalities was very common in these long-term cultures. All chromosomes were involved in these abnormalities. Loss of a morphologically normal X-chromosome may be related to the loss of estrogen receptors in these cell lines. With the exception of one subline, all cell lines had a short homogeneously staining region (HSR) on chromosome 7. Although the parent MCF-7 line had tetrahydrofolate dehydrogenase levels within the normal range, a lengthened HSR has been found in MCF-7 lines that are resistant to methotrexate. This observation strongly favors the association of an increased level of tetrahydrofolate dehydrogenase with HSR. In conclusion, the inherent genetic instability in this group of related cell lines may explain the heterogeneity found in this tumor. Continuous chromosomal rearrangements and numerical changes may reflect an ongoing process of selection and adaptation in these cell lines established from a breast carcinoma and may be characteristic of the aggressiveness of this neoplasm.

Cytogenetic studies in human T-cell lymphoma virus (HTLV)-positive leukemia-lymphoma in the United States.

Cytogenetic studies were conducted on fresh and cultured cells from 11 patients with human T-cell leukemia virus-associated adult T-cell leukemia-lymphoma. Clones with abnormal karyotypes were detected in 9 of the 11 patients. Chromosome numbers were near-diploid in cells from all but 1 patient who also had a tetraploid clone. The chromosome abnormalities in these cells were extensive; numerous complex structural changes were seen in every chromosome pair. Structural abnormalities occurred most frequently in chromosome 6. The 6 patients with chromosome 6 deletions had breakpoints at bands q11, q13, q16q23, q21q23, q22q24, and q23q24. The characteristic clinical features of these 6 patients were aggressive course, short survival, poor response to chemotherapy, high white blood cell counts, hypercalcemia, and bone lesions, whereas cytogenetically abnormal patients without chromosome 6q deletions tended to have a more indolent course. The precise role of the 6q deletion cannot be established with certainty from these data. However, this abnormality appears to occur with a greater than expected frequency in this large cell aggressive lymphoma, in association with hypercalcemia and lytic bone lesions.

Establishment of human mesothelioma cell lines (MS-1, -2) and production of a monoclonal antibody (anti-MS) with diagnostic and therapeutic potential.

We have established and characterized two mesothelioma cell lines, MS-1 and MS-2, in four attempts at long-term culture of these cells. Both MS-1 and MS-2 cells consistently express cytokeratin and vimentin, and both have long, slender microvilli. The cells that grew indefinitely (MS-1, MS-2) had a higher DNA index and a higher nucleus:cytoplasm ratio than did those cells that failed to grow (MS-3, MS-4). All mesothelioma cells, both in short- and in long-term culture, responded to phorbol ester induction by displaying morphological differentiation, such as an increase in the number of microvilli. The distribution of vimentin and cytokeratin in the cells, however, remained the same regardless of the growth pattern or the phorbol-ester treatment of the cells. We have used MS-1 cells to produce a monoclonal antibody (anti-MS) that reacts with mesothelioma cells, but rarely with reactive or normal mesothelial cells or with cells of normal tissues. The antibody does not induce the modulation of antigen, and it causes no direct or complement-mediated cytotoxicity of MS-1 or MS-2 cells. If a toxin or an isotope conjugate of the antibody is used, it may be valuable in the near future to test it for use in immunoimaging or immunotherapy.

Sister chromatid exchanges, chromosomal aberrations, and cytotoxicity produced by antitumor topoisomerase II inhibitors in sensitive (DC3F) and resistant (DC3F/9-OHE) Chinese hamster cells.

4'-(9-Acridinylamino)methanesulfon-m-anisidide, etoposide, and 2-methyl-9-hydroxyellipticinium are antitumor topoisomerase II (topo II) inhibitors. The relationship between drug-induced sister chromatid exchanges (SCEs) or chromosomal aberrations and cytotoxicity was investigated in Chinese hamster cells sensitive (DC3F) and resistant (DC3F/9-OHE) to topo II inhibitors. Thirty-min drug treatments produced SCEs and chromosomal aberrations in sensitive (DC3F) cells, 4'-(9-acridinylamino)methanesulfon-m-anisidide being more potent than etoposide or 2-methyl-9-hydroxyellipticinium at equimolar concentrations. Comparable treatments of resistant (DC3F/9-OHE) cells did not produce chromosomal damage. The cytotoxicity of 4'-(9-Acridinylamino)-methanesulfon-m-anisidide was also greater than that of etoposide or 2-methyl-9-hydroxyellipticinium in DC3F cells, and no cytotoxicity was observed in DC3F/9-OHE at drug concentrations that produced more than two logs of cell kill in DC3F cells. A plot of cytotoxicity versus SCEs showed a good correlation between the two parameters. Therefore, short treatments of mammalian cells with topo II inhibitors produce reversible topo II-mediated DNA breaks which are associated with chromosomal aberrations and SCEs whose number correlates with cytotoxicity. In addition, topo II mutant DC3F/9-OHE cells were more sensitive than DC3F cells to the chromosomal, DNA cross-linking and cytotoxic effects of mitomycin C and were equally sensitive to the cytotoxic effect of camptothecin.

Increased fragile sites and sister chromatid exchanges in bone marrow and peripheral blood of young cigarette smokers.

Cigarette smoking is considered to be the single most important acquired cause of cancer mortality. Studies of chromosome aberrations, sister chromatid exchanges, and fragile sites in peripheral blood or bone marrow are useful methods to detect the effects of the environmental mutagens or carcinogens found in cigarette smoke. The effects of smoking on the immature cells in the bone marrow have not been studied. Here, we examine the peripheral blood and bone marrow in 18 smokers (15 females and 3 males) with a median age of 25 years (range, 21-40) and an average cigarette use corresponding to 6 pack years. In both bone marrow cells and peripheral blood lymphocytes, we were able to show a significantly increased frequency of sister chromatid exchanges in smokers with a 5 or more cigarette pack year history, but not in those who smoked less than 5 pack years. We also found a higher frequency of sister chromatid exchanges in peripheral blood lymphocytes than in bone marrow cells. In addition, the peripheral lymphocytes of smokers demonstrated (a) a significantly higher frequency of fragile sites, (b) an increased number of metaphases with extensive breakage; and (c) elevated expression of fragile sites at the cancer breakpoints 3p14.2, 11q13.3, 22q12.2, and 11p13-p14.2 and at the oncogene sites bcl 1, erb B, erb A, and sis. Our results suggest that chromosomal DNA of peripheral blood lymphocytes is sensitive to cigarette smoking. Studies of the chromosomal changes in these cells provide an index of the mutagenic damage caused by these exogenous agents in individual patients and the ability of individuals to repair that damage, and might predict susceptibility to malignant events.

Correlations between intercalator-induced DNA strand breaks and sister chromatid exchanges, mutations, and cytotoxicity in Chinese hamster cells.

Intercalator-induced DNA strand breaks in mammalian cells represent topoisomerase II:DNA complexes trapped by intercalators. These complexes are detected as protein-associated DNA single-strand breaks (SSB) and DNA double-strand breaks (DSB) by filter elution. Using Chinese hamster lung fibroblasts (V79 cells) that were treated for 30 min with various concentrations of 4'-(9-acridinylamino)methanesulfon-m-anisidide or 5-iminodaunorubicin, we measured DNA strand breaks (SSB and DSB), sister chromatid exchanges (SCE), mutations at the hypoxanthine:guanine phosphoribosyltransferase locus, and cell killing. Further, we correlated DNA strand breakage with the three other parameters. Both drugs induced SCE, mutations, and cell killing at concentrations which also produced reversible DNA strand breaks. While the quantity of DSB correlated with SCE, mutations, and cytotoxicity for both drugs, we found more SCE, mutations, and cytotoxicity per SSB in cells treated with 5-iminodaunorubicin than in those treated with 4'-(9-acridinylamino)methanesulfon-m-anisidide. These data show that the DSB (but not the SSB) induced by 4'-(9-acridinylamino)methanesulfon-m-anisidide and 5-iminodaunorubicin at DNA topoisomerase II binding sites correlated closely with SCE, mutations, and cell killing and could therefore be responsible for their production.

Isolation of amplified and overexpressed DNA sequences from adriamycin-resistant human breast cancer cells.

An MCF-7 human breast cancer cell line was selected which was 200-fold more resistant to Adriamycin than the wild type cell line. This Adriamycin-resistant (AdrR) cell line exhibited a multidrug-resistant phenotype and was cross-resistant to a wide range of antineoplastic agents including Vinca alkaloids, anthracyclines, and epipodophyllotoxins. Cytogenetic analysis of the AdrR cell line showed the presence of homogeneously staining regions on several chromosomes which were not present in the parental cell line. Using the technique of in-gel renaturation, DNA sequences which were amplified 50- to 100-fold in the AdrR cell line and which covered a total of over 140 kilobases were isolated. In addition, AdrR cells were found to contain amplified and overexpressed sequences which were homologous to hamster P-glycoprotein gene sequences. A hamster cDNA P-glycoprotein gene probe was used to screen a lambda gt10 cDNA library made from human AdrR cell line mRNA and human cDNA sequences homologous to the P-glycoprotein gene were isolated. Hybridization studies with the cloned human cDNA (pADR1) showed that the AdrR MCF-7 cell line contained a 60-fold amplification of this DNA sequence and that polyadenylated mRNA from the AdrR cell line contained a 4.8-kilobase transcript which was overexpressed 45-fold. There was a direct correlation between DNA and RNA copy number of this sequence and level of resistance among several MCF-7 Adriamycin-resistant cell lines. In situ hybridization studies demonstrated that the human P-glycoprotein gene sequence was found on chromosome 7q21.1 in normal human lymphocytes and that amplified DNA sequences isolated from the AdrR MCF-7 cells by the in-gel hybridization technique were linked to the human P-glycoprotein sequences in the homogeneously staining regions in the AdrR cells.

A novel method for selection of invasive tumor cells: derivation and characterization of highly metastatic K1735 melanoma cell lines based on in vitro and in vivo invasive capacity.

Tumor cell invasion of basement membranes is required at several steps in the process of metastasis. To study the genetic and biochemical events mediating invasion, a variant cell line (TK) was selected from the metastatic M2 K1735 murine melanoma cell line. A novel selection procedure was used, based on in vitro and in vivo invasion and growth upon basement membrane and stroma. Additionally, two extrapulmonary metastases of the TK cell line, TK-Eve and TK-Liver, were established as cell lines and characterized. The TK cell line demonstrates greater metastatic potential in vivo and invasive ability in vitro than the parent M2 cell line, confirming the validity of the selection procedure. In addition, the M2 and TK cell lines were examined for other cell functions involved in the metastatic process. Cellular growth rates and sensitivity to T lymphocyte and natural killer cell lysis were not determining factors in the metastatic potentials of the M2 and selected cell lines; possible macrophage contribution to metastatic behavior was noted. [35S]methionine pulse labeling of protein synthesis and karyotypic analysis confirm the close relationship of parental and selected cell lines.

Translocation of c-sis protooncogene in peripheral neuroepithelioma.

Molecular genetic analysis of the c-sis protooncogene was performed on two neuroepithelioma cell lines carrying a t(11;22)(q24;q12). The c-sis protooncogene was found by in situ hybridization to be translocated from its germline position on chromosome #22 to the derivative chromosome #11 in each cell line. However, it was not rearranged or amplified in either cell line examined. In addition, we did not detect c-sis transcripts in Northern blots of poly (A)+ RNA. This is similar to results found in Ewing's sarcoma, which carries a cytogenetically indistinguishable translocation from neuroepithelioma.

A nonrandom chromosomal abnormality, del 3p(14-23), in human small cell lung cancer (SCLC).

In order to determine whether or not there are specific chromosomal changes in small cell lung cancer (SCLC), karyotypic analyses of 16 continuous SCLC tissue culture lines, three fresh tumor specimens (bone marrow), one direct preparation of bone marrow involved with SCLC, and two lymphoblastoid lines derived from SCLC patients were studied. Cell lines were derived from primary tumor, or metastases to bone marrow, subcutaneous nodules, or pleural fluid; all 16 lines had biochemical and histologic properties characteristic of SCLC. Of the 15 males and 3 females, 6 patients had no prior treatment. All of the 16 cell lines, the 3 fresh specimens, and the direct bone marrow preparation had a common deletion of the short arm of chromosome #3. Use of the shortest region of overlap analysis showed the common deletion was of the short arm in the regions p(14-23). This specific chromosomal abnormality, del 3p, was not found in five non-SCLC cell lines studied and is of major potential biological and diagnostic importance.

Ring chromosome in a case of acute myelomonocytic leukemia: its significance and a review of the literature.

The presence of ring chromosomes is a rare finding in patients with de novo acute nonlymphocytic leukemia (ANLL). In this report we describe the cytogenetic abnormalities, including ring chromosomes, of a 45-year-old white male who had ANLL of the myelomonocytic type (M4). All of the leukemic cells had both a small unidentified ring and a medium-sized ring which was derived from chromosomes 2 and 11, r(2;11)(2p25;11p15q23;2q37). Four different types of rings or other structural abnormalities were derived from this medium-sized ring. A review of the literature indicated that the median survival of ANLL patients with either identified or unidentified ring chromosomes was 5 to 6 months, and that the presence of these cytogenetic abnormalities was related to a poor prognosis.

Cytogenetic effects of amsacrine on human lymphocytes in vivo and in vitro.

Amsacrine (m-AMSA) is presently being utilized in phase I-II studies at the Medicine Branch, National Cancer Institute, National Institutes of Health (Bethesda, MD), and is being administered as a continuous infusion to patients with progressive malignancy after conventional therapy. In the present study, we examined the effects of this drug, in vivo and in vitro, on chromosomal morphology and the frequency of sister chromatid exchange (SCE) induction in human peripheral blood lymphocytes. In the in vivo studies, eight patients receiving 30 mg/m2/day of m-AMSA by continuous infusion showed increased levels of chromosomal aberrations, up to a maximum of 14% (median; range, 10%-24%) at 96 hours compared to 1% (median; range, 0%-4%) in the control group; no increase was noted in SCE frequencies. In vitro studies of normal human lymphocytes with various concentrations of m-AMSA, including doses comparable to those used in vivo, showed both increased levels of chromosomal aberrations, ranging from 8% to 100%, and increased SCEs, ranging from 1.5 times the normal at the lowest concentration studied (0.005 microgram/ml) to 12 times the normal (0.25 microgram/ml). In correlating SCEs with breaks in the in vitro studies, it was found that two-thirds of the total breaks occurred at SCE points; this indicates a close relationship between the events leading to these two types of aberrations. In vitro isotope incorporation studies showed inhibition of both RNA and DNA syntheses but no significant inhibition of protein synthesis; at a dose of 5.0 micrograms/ml, RNA synthesis was 80% inhibited and DNA synthesis was 60% inhibited after 36 hours of incubation with m-AMSA. The difference in the results between in vitro and in vivo SCE studies may be due to possible DNA repair in the in vivo-treated lymphocytes in m-AMSA-free medium in vitro, a process not available to the in vitro-treated cells.

Human glutathione S-transferases. The Ha multigene family encodes products of different but overlapping substrate specificities.

The human glutathione S-transferase cDNAs encoding subunits 1 and 2 contain intrinsic ribosome-binding sites in their 5'-untranslated regions for direct expression in Escherichia coli. We show that functional human GSH S-transferases 1-1 and 2-2 are synthesized from lambda gt11 cDNA clones lambda GTH1 and lambda GTH2 in phage lysates of E. coli Y1090, in lysogens of E. coli Y1089, and from the plasmid expression constructs in pKK223-3. The E. coli-expressed human GHS S-transferases 1-1 and 2-2 do not have blocked N termini in contrast to those directly purified from human livers. These two isozymes, with 11 amino acid substitutions between them, are similar in their Km values for GSH and 1-chloro-2,4-dinitrobenzene and Kcat values for this conjugation reaction. The human GSH S-transferase 2-2, however, is a more active GSH peroxidase than transferase 1-1 toward cumene hydroperoxide and t-butyl hydroperoxide. Our results indicate that different members of a GSH S-transferase gene family with limited amino acid substitutions have different with limited amino acid substitutions have different but overlapping substrate specificities. We propose that accumulation of single amino acid replacements may be an important mechanism for generating diversity in GSH S-transferases with various xenobiotic substrates. In situ chromosomal hybridization results show that the GSH transferase Ha genes are located in the region of 6p12.