Biomedical subjects

M C Hollander

Publications and source records attributed to M C Hollander.

26 records · Page 2Linked to original sources

PubMed1990

Expression of the O6-methylguanine-DNA methyltransferase gene MGMT in MER+ and MER- human tumor cells.

DNA probes prepared from human O6-methylguanine-DNA methyltransferase complementary DNA were hybridized to mRNA isolated from human liver and fifteen human tumor cell lines proficient (Mer+) or deficient (Mer-) in transferase activity. Liver and Mer+ cells contained levels of transferase-specific mRNA that correlated with their transferase activity levels, whereas Mer- cells contained undetectable amounts of transferase mRNA. The mRNA levels were not induced in human cells by treatments that induce other DNA damage-inducible genes. These results demonstrate that in human cells the transferase gene is constitutively expressed, that its expression is related to activity levels, and that in Mer- tumor cells the expression of the transferase gene is probably blocked at the level of mRNA production.

Cell Line↗

PubMed1990

Estimation of relative mRNA content by filter hybridization to a polythymidylate probe.

We have developed an accurate and sensitive method for the quantitation of relative mRNA in RNA samples. This procedure is especially applicable for normalizing numerous RNA samples which are to be analyzed by dot blot hybridization. The method entails hybridization of a polythymidylate probe with RNA bound to filters. The relative hybridization of polythymidylate probe to RNA is proportional to the polyadenylate RNA content of the RNA samples. Hybridization of polythymidylate probe to RNA is not dependent on any cell treatment or growth condition that we have observed, and experimental variation is minimized. Therefore, the relative hybridization to polythymidylate probe is a better method for standardizing the amount of mRNA in RNA samples than is relative hybridization to cDNA probes such as actin or beta 2-microglobulin, whose transcript levels may vary according to cell treatment. Furthermore, since experimental variation and errors are minimized, this procedure is suitable for normalizing RNA samples in which there are only small changes in the levels of particular transcripts.

Animals↗

PubMed1989

Induction of fos RNA by DNA-damaging agents.

We have found that a wide variety of DNA-damaging agents and heat shock increase fos RNA to different extents in Chinese hamster ovary cells. These include the monofunctional alkylating agents methylmethane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine, the cross-linking agents cis-Pt(II) diamminedichloride and mechlorethamine HCl, the DNA base-damaging agents 4-nitroquinoline-N-oxide and N-acetoxy-2-acetylaminofluorene, ultraviolet and near-ultraviolet radiation, hydrogen peroxide, and Adriamycin. Prolonged increases in fos RNA were observed, with persistence to 16 h following treatment. Variation in the amount of fos RNA induction by these agents and the diverse types and frequencies of cellular damage produced by them suggested that there might be several different mechanisms responsible for increased fos RNA.

Animals↗

PubMed1989

Ubiquitin mRNA is a major stress-induced transcript in mammalian cells.

Ubiquitin mRNA was found to be an abundant transcript which was induced by heat shock (HS), and certain other stresses in mammalian cells. In Chinese hamster cells, the 2 major ubiquitin transcripts of 2.6 kb and 1.7 kb were induced coordinately, while a minor ubiquitin transcript of 0.8 kb was not induced; the response was similar in human cells with induction of the 2.5 kb Ub C and 1.0 kb Ub B transcripts. A representative ubiquitin cDNA clone, isolated from a cDNA library derived from HS-treated Chinese hamster cells, coded for a typical tandem repeat polyubiquitin transcript. Only a portion of the 5' nontranslated sequence of this clone had homology with the previously published corresponding region in human Ub B mRNA. Oligonucleotide probes complementary to the portion of the 5' nontranslated sequence with homology to the human sequence and also portions with no homology hybridized only to the 1.7 kb transcript. There was coordinate induction of ubiquitin, HSP27, and HSP70 mRNA by HS as determined by both increased RNA and increased transcription. Ubiquitin mRNA was induced by certain DNA damaging agents, in particular the alkylating agent methylmethane sulfonate, or incubation in isoleucine-deficient medium under conditions where the other HSP mRNA were not.

Amino Acid Sequence↗

PubMed1989

Induction of heat shock protein transcripts and B2 transcripts by various stresses in Chinese hamster cells.

We have investigated the induction of known hsp (heat shock protein) RNA and other heat shock (HS) inducible transcripts in Chinese hamster cells by various stresses including DNA damaging agents. cDNA clones coding for at least 14 different HS-inducible transcripts were isolated. By DNA sequence analysis and homology with cDNA clones of other species, some of these cDNA clones were identified as coding for hsp27, hsp89 alpha, hsp89 beta, two different hsp70s, ubiquitin, and the HS-inducible RNA polymerase III transcript B2. In addition, hsp-related cDNA clones, hsp60 and four with hsp70 homology, were isolated which coded for transcripts which were not induced by HS or other stresses in two different Chinese hamster cell lines. After HS or treatment with the HS-mimetic agent ethanol, there was coordinate induction of all 14 transcripts. With severe HS treatments which produced substantial cytotoxicity, the increase in all transcripts except B2 RNA was delayed and, in some cases, suppressed. The only DNA damaging agent, which induced many HS-inducible transcripts, was high-dose methylmethane sulfonate (MMS). However, induction by MMS was not coordinate for all transcripts as it was for HS, and B2 RNA was not induced. hsp27 RNA induction differed from the others in several respects including induction by irradiation and other agents which produce high levels of DNA damage repaired by nucleotide excision repair. The implications of these findings in cellular events such as cytotoxicity, thermotolerance, and regulation of stress responses will be discussed.

Animals↗

PubMed1989

Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents.

More than 20 different cDNA clones encoding DNA-damage-inducible transcripts in rodent cells have recently been isolated by hybridization subtraction (A. J. Fornace, Jr., I. Alamo, Jr., and M. C. Hollander, Proc. Natl. Acad. Sci. USA 85:8800-8804, 1988). In most cells, one effect of DNA damage is the transient inhibition of DNA synthesis and cell growth. We now show that five of our clones encode transcripts that are increased by other growth cessation signals: growth arrest by serum reduction, medium depletion, contact inhibition, or a 24-h exposure to hydroxyurea. The genes coding for these transcripts have been designated gadd (growth arrest and DNA damage inducible). Two of the gadd cDNA clones were found to hybridize at high stringency to transcripts from human cells that were induced after growth cessation signals or treatment with DNA-damaging agents, which indicates that these responses have been conserved during mammalian evolution. In contrast to results with growth-arrested cells that still had the capacity to grow after removal of the growth arrest conditions, no induction occurred in HL60 cells when growth arrest was produced by terminal differentiation, indicating that only certain kinds of growth cessation signals induce these genes. All of our experiments suggest that the gadd genes are coordinately regulated: the kinetics of induction for all five transcripts were similar; in addition, overexpression of gadd genes was found in homozygous deletion c14CoS/c14CoS mice that are missing a small portion of chromosome 7, suggesting that a trans-acting factor encoded by a gene in this deleted portion is a negative effector of the gadd genes. The gadd genes may represent part of a novel regulatory pathway involved in the negative control of mammalian cell growth.

Amino Acid Sequence↗

PubMed1989

Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells.

Only a few of the genes involved in DNA repair in mammalian cells have been isolated, and induction of a DNA repair gene in response to DNA damage has not yet been established. DNA polymerase beta (beta-polymerase) appears to have a synthetic role in DNA repair after certain types of DNA damage. Here we show that the level of beta-polymerase mRNA is increased in CHO cells after treatment with several DNA-damaging agents.

Animals↗

PubMed1988

DNA damage-inducible transcripts in mammalian cells.

Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury.

Acetoxyacetylaminofluorene↗