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Biomedical subjects

R Sager

Publications and source records attributed to R Sager.

At least 91 records · Page 5Linked to original sources
PubMed1989

Spontaneous mutation rates of tumorigenic and nontumorigenic Chinese hamster embryo fibroblast cell lines.

The genomic stability of a series of nontumorigenic, tumorigenic, and tumor-derived Chinese hamster embryo fibroblastic (CHEF) cell lines was compared by examining their rates of spontaneous mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, using thioguanine resistance for selection of mutants. The spontaneous mutation rates were 1.1 x 10(-6) mutations/cell/generation in the non-tumor-forming CHEF/18 cell line and 4.9 x 10(-6) in the tumorigenic CHEF/16 cells. Three tumorigenic and tumor-derived CHEF cell lines derived from CHEF/18 (J132 3-2 T3L, focus 2, focus 3) and two lines (16-2 Tuk 4 and 204 Bu50 Tuk 2) derived from CHEF/16 were chosen on the basis of their karyotypes, which demonstrated a considerable level of chromosomal rearrangement. Mutation rates of four of these five lines ranged from 1.2 x 10(-6) to 8.9 x 10(-6) mutations per cell per generation. Only the fifth line, 16-2 Tuk 4, showed a significantly elevated rate of mutation as compared with the nontumorigenic CHEF/18 cell line. Thus, we have found no simple correlation between spontaneous mutation rate and the malignant phenotype, and we conclude that mutation rate per se is not a sensitive index of malignancy. In addition, we have compared three methods of calculating mutation rate and find that they rank the cell lines in the same order, but each stresses a different aspect of the distribution and therefore produces different estimates of the mutation rate.

Animals↗
PubMed1989

A newly established metastatic breast tumor cell line with integrated amplified copies of ERBB2 and double minute chromosomes.

A continuous line of human mammary tumor cells, called 21MT, has been established in culture from a pleural effusion of a 36-year-old woman with metastatic breast cancer. The cells are epithelial as shown by morphology and expression of keratins and are mammary tumor cells as shown by expression of the HMFG-2 antigenic determinant. The cells grow well both in DFCI-1, a partially defined medium containing pituitary extract and 1% fetal bovine serum, and in alpha-minimum essential medium (alpha-MEM) supplemented with 10% serum, epidermal growth factor (EGF), insulin, and hydrocortisone. Karyotypic analysis of cells at early passage has shown the presence of rearranged (marker) chromosomes as well as aneuploidy with a net DNA content in the tetraploid range, confirmed by DNA cytofluorography, as well as double minute chromosomes in about 5% of the cells. Southern blots have revealed a 40-fold amplification of the ERBB2 gene and a 50-fold overexpression of its mRNA. The amplification of ERBB2 DNA was localized by in situ hybridization to one of the marker chromosomes but not to the double minutes. It is inferred, therefore, that at least two genes have been amplified in these cells.

Adult↗
PubMed1989

Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types.

A medium is described that supports long-term growth in culture of human primary mammary tumor cells, of normal epithelial cells from mammoplasty, and of mammary tumor cell lines. Tumor cells are shown to be distinguishable from normal mammary epithelial cells by morphology, by growth requirements, and by two markers: preferential expression of the HMFG-2 epitope on tumor cells and preferential retention in tumor cell mitochondria of the lipophilic fluorescent dye rhodamine 123. Differential fluorescence of HMFG-2 fluorescein-conjugated antibodies can be used as a basis for separation of normal and tumor cells in a fluorescence-activated cell sorter, as can differential retention of rhodamine 123.

Breast↗
PubMed1989

High frequency of large spontaneous deletions of DNA in tumor-derived CHEF cells.

Spontaneous mutations arising at the HPRT locus were examined in 126 mutants recovered from a series of six CHEF-derived cell lines. Altered restriction fragment patterns were characterized by Southern blot hybridization, and gene expression by RNA blot hybridization. Point mutants and gene-expression mutants predominated in the control (nontumorigenic) 18-1D-3 cell line and in two tumor-derived lines, one of which (16-2 Tuk 4) displayed a mutator phenotype. In the other three lines, the majority of mutants had large partial or whole gene deletions. These results suggest that mutant enzymes in DNA replication or repair play an important role in neoplastic progression by causing extensive deletions in DNA, including excision of genes that encode tumor-suppressor functions, and deletion of regulatory sequences in protooncogenes.

Blotting, Southern↗
PubMed1988

Expression of growth factors and oncogenes in normal and tumor-derived human mammary epithelial cells.

The expression of genes which may be involved in the regulation of human mammary epithelial cell growth [transforming growth factors alpha and beta] and tumorigenesis [c-myc, erbB2, epidermal growth factor receptor (EGFR), Ha-ras, pS2] has been compared in similarly cultured normal cell strains and tumor cell lines. We have found that the normal breast cells produce high levels of EGFR mRNA, which are translated into nearly 10(5) low affinity epidermal growth factor-binding molecules/cell. In the estrogen receptor-negative lines examined, the EGFR gene was expressed at levels comparable to those in the normal cells. In contrast, EGFR and transforming growth factor alpha mRNAs were reduced in estrogen receptor-positive tumor lines compared to estrogen receptor-negative lines and normal cells. Steady state mRNA levels for transforming growth factor beta, erbB2, c-myc, and Ha-ras in the normal cells were greater than or comparable to those in all of the breast tumor lines. Furthermore, in the absence of gene amplification, only one of the genes examined (i.e., pS2) was overexpressed in a subset of the tumor cells compared to their normal counterparts. Several reports by other investigators have described overexpression of some of these genes in breast biopsies and in tumor lines in studies lacking normal controls. Thus, our results, in which the same genes were not overexpressed compared to normal cells unless amplified, underscore the importance of including appropriate normal controls in studies aimed at defining aberrant patterns of gene expression in tumor cells.

Breast↗
PubMed1988

Detection of heavy methylation in human repetitive DNA subsets by a monoclonal antibody against 5-methylcytosine.

A monoclonal antibody (IgM) against 5-methylcytosine (mC) was isolated and characterized. It showed a high specificity for mC with a cross-reactivity of less than 1% with cytosine and 0.1% with thymidine. An improved immunohybridization method, originally developed with polyclonal antibodies (Sano et al. (1980) Proc. Natl. Acad. Sci. USA 77, 3581), was applied to detect mC in immobilized DNA using the new monoclonal preparation. Human genomic DNA was cleaved with the restriction enzyme EcoRI and successively fractionated by malachite-green affinity chromatography and agarose gel electrophoresis. The fractionated DNA was transferred to nitrocellulose paper and treated with the anti-mC monoclonal antibody. Heavy methylation was observed in EcoRI-ladders of repetitive sequences of 1360, 1750, 2200 and 3400 bp, while 340, 660 and 2700 bp fragments were less methylated. The results show that methylation occurs in limited subsets of satellite II and III repetitive DNAs that contain high amounts of methylatable CpG dinucleotides, or CpG clusters.

5-Methylcytosine↗
PubMed1988

Genetic analysis of tumorigenesis. XXXI: Retention of short arm of chromosome 3 in suppressed CHEF cell hybrids containing c-Ha-ras (EJ) gene.

Hybrids between nontransformed Chinese hamster embryo fibroblast (CHEF) cells and their c-Ha-ras (EJ) -transformed derivatives are suppressed for tumor-forming ability when tested at early passage. Hybrid subclones with suppressed (fibroblastic) or transformed appearance have now been selected by multiple recloning. Morphology, but not serum or anchorage requirement, was a sensitive indicator of suppression: Subclones with normal morphology were nontumorigenic, subclones with transformed morphology were highly tumorigenic, and intermediate subclones (7-70% normal colonies) formed tumors with a frequency of 17-50%. Suppressed lines retained the short arm of chromosome 3, but transformed and tumor-derived lines had lost this region (greater than or equal to 1 copy). Transformed and tumor-derived cells exhibited additional chromosome changes, including the loss of at least one copy of chromosomes 7 and/or 8. These findings suggest that a tumor suppressor gene lies on the short arm of chromosome 3, consistent with prior studies from this laboratory. Other suppressor genes may be located on chromosomes 7 and 8.

Animals↗
PubMed1988

Genetic analysis of tumorigenesis: XXXII. Localization of constitutionally amplified KRAS sequences to Chinese hamster chromosomes X and Y by in situ hybridization.

The KRAS gene is constitutionally amplified in the Chinese hamster. We have mapped the amplified sequences by in situ hybridization to two major sites on the X and Y chromosomes, Xq4 and Yp2. No autosomal site was detected despite a search under relaxed hybridization conditions. KRAS DNA is amplified about 50-fold compared to a human cell line known to have a diploid number of KRAS sequences, whereas mRNA expression is 5- to 10-fold lower than in normal human cells. While mRNA expression levels do not necessarily parallel gene copy number, the low expression level strongly suggests that the amplified sequences are transcriptionally silent. It is suggested that the amplified sequences arose from the original KRAS gene on chromosome 8 and that the KRAS sequences on the Y chromosome arose by X-Y recombination.

Animals↗
PubMed1988

Functional diversity of gro gene expression in human fibroblasts and mammary epithelial cells.

Previous studies of gro and related genes that are overexpressed in transformed fibroblasts suggest that gro may encode a specific growth regulator. However, DNA and protein sequence comparisons reveal relatedness to platelet factor 4 and other proteins involved in the inflammatory response. In this paper, both growth-related and cytokine-induced responses in gro gene expression are described. Human foreskin fibroblasts are shown to express approximately 10-fold elevated gro, myc, and fos mRNAs in response to serum and to phorbol 12-myristate 13-acetate stimulation, with early response kinetics indicative of growth regulation. In response to interleukin 1, however, in growing cells gro mRNA is elevated at least 100-fold but myc remains constant and fos is not expressed, suggesting a second regulatory pathway. In normal cultured mammary epithelial cells, gro is constitutively expressed, and elevated mRNA levels are induced by phorbol 12-myristate 13-acetate, but not by interleukin 1. However, most carcinoma cell lines examined do not express gro mRNA, suggesting a third function of gro as a negative growth regulator in epithelial cells.

Chromosome Banding↗
PubMed1988

Mutation rates and mutational spectra in tumorigenic cell lines.

The rate and molecular nature of spontaneous mutations at the hgprt locus were examined in a series of tumorigenic and non-tumorigenic closely related Chinese hamster fibroblastic (CHEF) cell lines. Mutation rates of tumorigenic cells determined by fluctuation analysis were found to range from the low rate also seen in non-tumorigenic cells to values increased up to 25-fold. No simple correlation was found between elevated mutation rates and tumorigenic potential. The nature of the mutational event was examined in a set of 136 thioguanine resistant mutants selected from several tumorigenic and non-tumorigenic CHEF lines. Significantly different frequencies of point mutations were found compared with large partial or whole gene deletions in different cell lines. The clonal inheritance of specific mutational patterns as well as the high frequencies of large deletions were novel findings. Hypotheses to explain these results are discussed in relation to the known genomic instability of tumour cells. I am honoured by the opportunity to contribute to this collection of papers dedicated to Professor Guido Pontecorvo on the occasion of his eightieth birthday. For almost half of this time I have been privileged to enjoy his friendship, and to benefit from his influence at critical stages in my scientific development.

Animals↗
PubMed1987

Transfection with plasmid pSV2gptEJ induces chromosome rearrangements in CHEF cells.

In previous cytogenetic studies, trisomy for 3q was found to be the most frequent chromosome change associated with induced tumorigenicity by a variety of agents in Chinese hamster cells. Here we describe similar chromosome changes in 11 lines of CHEF/18 cells transfected with the mutant c-Ha-ras containing plasmid pSV2gptEJ. All 11 lines contained the transfected EJ gene and expressed increased levels of p21, the EJ gene product. Ten of the 11 lines were tumorigenic and all but 2 of these were trisomic for all or part of 3q. One line remained diploid and was nontumorigenic despite expressing elevated p21. Two tumorigenic lines from "hit-and-run" transfection with pSV2gpt were shown to express only control levels of p21, but they were trisomic for 3q. These results show that increased p21 expression is neither necessary nor sufficient for inducing tumorigenicity of CHEF cells. We propose that tumorigenicity in the transfected CHEF/18 cells of this study was induced by chromosome rearrangements, especially trisomy for 3q, that occurred at increased frequencies following transfection with pSV2gptEJ.

Animals↗
PubMed1987

Constitutive overexpression of a growth-regulated gene in transformed Chinese hamster and human cells.

Comparison by subtractive hybridization of mRNAs revealed a moderately abundant message in highly tumorigenic CHEF/16 cells present at very low levels in closely related nontumorigenic CHEF/18 cells. After cloning and sequencing the corresponding cDNA, computer comparison showed closest homology with the human connective tissue-activating peptide III (CTAP III). The human tumor cell cDNA hybridizing with the Chinese hamster clone was isolated, sequenced, and found to have closer similarity to the Chinese hamster gene than to CTAP III. Thus, the cloned cDNAs from Chinese hamster and human cells represent a different gene, named gro. Studies of its transcriptional regulation have shown that expression is tightly regulated by growth status in normal Chinese hamster and human cells and relaxed in the tumorigenic cells so far examined.

Amino Acid Sequence↗
PubMed1987

Genetic analysis of tumorigenesis: a conserved region in the human and Chinese hamster genomes contains genetically identified tumor-suppressor genes.

Regional chromosome homologies were found in a comparison of human 11p with Chinese hamster 3p. By use of probes that recognize six genes of human 11p (INS, CAT, HBBC, CALC, PTH, and HRAS), the corresponding genes were localized by in situ hybridization on Chinese hamster chromosome 3. INS and CAT were located close to the centromere on 3p, whereas HBBC, CALC, and PTH were at 3q3-4 and HRAS at 3q4. Extensive prior data from chromosome studies of tumorigenic and tumor-derived Chinese hamster cells have suggested the presence of a tumor-suppressor gene on 3p. Two tumor-suppressor genes have been described on human 11p, one linked to CAT and one to INS. The present study raises the possibility that the Chinese hamster suppressor may be closely linked to INS or CAT.

Animals↗
PubMed1986

An endonuclease from Chlamydomonas reinhardtii that cleaves the sequence TATA.

An endodeoxyribonuclease, designated CreI, was purified 16,000-fold from zygotes of the eukaryote Chlamydomonas reinhardtii. CreI preferentially attacks the sequence TATA producing double strand breaks with 3'-phosphomonoester and 5'-hydroxyl termini. The endonuclease has an Mr = 27,000 and requires Ca2+ at pH 7.5 for optimal activity.

Base Sequence↗
PubMed1986

Suppression of tumor growth by senescence in virally transformed human fibroblasts.

Normal human cells whether embryonic, neonatal, or adult are resistant to experimentally induced tumorigenesis in contrast to rodent or chicken cells. We showed previously that neither transformation with simian virus 40 DNA nor transfection with human mutant HRAS DNA immortalized FS-2 cells (diploid, neonatal human fibroblasts). Further, tumorigenicity was not induced, despite expression of the respective transforming gene products tumor (T) antigen or p21. Here we describe treatment of FS-2 and FSSV cells with baboon endogenous virus pseudotyped Kirsten murine sarcoma virus. FSSV cells were derived from individual foci of simian virus 40-transformed FS-2 cells. The retrovirus-treated FS-2 cells (called FSK) appeared heavily granulated and expressed viral p21 but senesced during passage in culture and were not tumorigenic. The retrovirus-treated FSSV-27 cells (called FSVK-27) expressed simian virus 40 tumor antigen, had elevated levels of viral p21 protein, and formed transient tumors in nude mice. Whether grown in culture or explanted from small tumors, the FSVK-27 cells senesced. The FSVK-46 cells senesced before tumor growth occurred. On the contrary, Kirsten murine sarcoma virus (baboon endogenous virus) treatment of immortalized nontumorigenic human fibroblasts expressing simian virus 40 tumor antigen (Va2 cells) led to consistent tumor formation. The results illustrate the importance of senescence in restricting the tumor-forming ability of human cells.

Animals↗
PubMed1985

Genetic analysis of tumorigenesis: XXI. Suppressor genes in CHEF cells.

In previous studies, fusions of transformed X nontransformed CHEF cells have produced hybrids that were suppressed for transformed traits and for tumor formation. During subsequent growth, the suppressed phenotypes were lost coincident with chromosome loss, and in one study the loss of anchorage dependence was correlated with loss of chromosome 1. In this paper, suppression of serum and anchorage requirements for growth is examined with the use of double-mutant tester stocks. Nontumorigenic low serum mutants from CHEF/18 cells are shown to complement with the lowered serum requirement of CHEF/16, a tumorigenic line, indicating that the high serum requirement is dominant and regulated by at least two genes. Similar results were previously reported for the anchorage requirement. Suppression of the two traits is found to segregate independently in hybrid subclones with reduced chromosome numbers, showing that different genes control suppression of the serum and anchorage requirements. Evidence for two modes of suppression, by dominant alleles of transformation genes and by unrelated genes, is presented and discussed.

Animals↗