Biomedical subjects

R S Day

Publications and source records attributed to R S Day.

At least 37 records · Page 2Linked to original sources

PubMed1995

Site specificity of incisions at G:T and O6-methylguanine:T base mismatches in DNA by human cell-free extracts.

Cell-free extract from human tumor cell line A1235 (lacking O6-methylguanine-DNA methyltransferase) was employed to compare incision at G:T base mispairs with that at O6-methylguanine (m6G):T pairs at two different sites (sites 20 and 25) in 45-bp heteroduplexes. To study the effect of neighboring bases on the activity(ies), the base pair immediately 5' to the mismatched G at each site was varied to provide four contexts: CpG:T, TpG:T, ApG:T, and GpG:T (and two analogous series for m6G:T pairs). At site 20, cell-free extract produced observable incision only in the 45-bp DNA with the G:T mispair in the CpG:T context, giving a product with incisions immediately 5' and 3' to the mismatched T. We observed incision of neither the strand containing the mismatched G nor the DNAs with the site 20 ApG:T, GpG:T, and TpG:T mismatches. By contrast, at site 25, incision specificity was different. All four G:T mismatched DNAs were incised, and the ApG:T-25, GpG:T-25, and TpG:T-25 DNAs were incised 1-3 bonds 3' to the mismatched T, while similar in other respects to the CpG:T-25 DNA, which showed a pattern like the CpG:T-20 DNA. CpG:T-20 specific incision activity in the extract was strongly inhibited by both CpG:T (sites 20 and 25) DNAs, but at least 10-fold more poorly by DNAs with Apg:T-25 and GpG:T-25 pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗

PubMed1995

Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line.

The radiosensitive rodent mutant cell line xrs-5 is defective in DNA double-strand break repair and lacks the Ku component of the DNA-activated protein kinase, DNA-PK. Here radiosensitive human cell lines were analyzed for DNA-PK activity and for the presence of related proteins. The radiosensitive human malignant glioma M059J cell line was found to be defective in DNA double-strand break repair, but fails to express the p350 subunit of DNA-PK. These results suggest that DNA-PK kinase activity is involved in DNA double-strand break repair.

Amino Acid Sequence↗

PubMed1995

Modification of the radiosensitivity of human cells to which simian virus 40 T-antigen was transfected.

Effects of the introduction of the Simian virus 40 T-antigen (SV40 T-Ag) gene to cultured human cells were examined in relation to radiosensitivity. Two relatively radioresistant tumor cell lines (T98 and G361) became significantly radiosensitive after the introduction of SV40 T-Ag, whereas radiosensitive tumor cell lines did not show a change in radiosensitivity. In contrast, a human fibroblast cell line became radioresistant after SV40 T-Ag introduction. T98 cells which have a mutation at codon 237 in the p53 gene were unable to form a complex between p53 protein and SV40 T-Ag, whereas G361, which became radiosensitive by a SV40 T-Ag introduction, formed the complex. This indicates that the status of p53 is independent of the change in radiosensitivity in the cell lines studied.

Antigens, Polyomavirus Transforming↗

PubMed1995

Radiation-induced DNA damage and repair in cells of a radiosensitive human malignant glioma cell line.

The induction and repair of DNA double-strand breaks were studied in cells of two isogenic human malignant glioma cell lines which vary in their SF2 values by a factor of approximately 30. M059J cells are radiosensitive (SF2 = 0.02) and lack the p350 component of DNA-dependent protein kinase (DNA-PK); M059K cells are radioresistant (SF2 = 0.64) and express normal levels of DNA-PK. Zero integrated field gel electrophoresis and alkaline sucrose gradient experiments indicated that equivalent numbers of DNA lesions were produced by ionizing radiation in M059J and M059K cells. To compare the capacity of both lines to repair sublethal damage, the split-dose recovery experiment after exposure to equitoxic doses of radiation was carried out. Significant sublethal damage repair was shown for M059K cells, with a 5.8-fold increase in relative survival peaking at 4 h, whereas M059J cells showed little repair activity. Electrophoresis studies indicated that more double-strand breaks were repaired by 30 min in M059K cells than in M059J cells. These results suggest that deficient DNA repair processes may be a major determinant of radiosensitivity in M059J cells.

Cell Line↗

PubMed1994

CDK4 amplification is an alternative mechanism to p16 gene homozygous deletion in glioma cell lines.

Recently, it has been shown that a gene encoding the cyclin-dependent kinase 4 inhibitory protein, p16, is frequently targeted for homozygous deletions in several types of tumor cell lines, including those established from malignant gliomas. Here we have examined 32 glioma cell lines for amplification-associated overexpression of the CDK4 gene as an alternative mechanism for abrogating the growth-regulatory effects of p16. Two of the cell lines revealed high-level expression of CDK4 in association with gene amplification, and this alteration was observed among the 10 cases having intact p16 genes. Consequently, 24 of 32 glioma cell lines revealed one of two alternative genetic alterations, each of which indicates that increased cdk4 kinase activity is important to glial tumor development.

Base Sequence↗

PubMed1994

A cell cycle regulator potentially involved in genesis of many tumor types.

A putative tumor suppressor locus on the short arm of human chromosome 9 has been localized to a region of less than 40 kilobases by means of homozygous deletions in melanoma cell lines. This region contained a gene, Multiple Tumor Suppressor 1 (MTS1), that encodes a previously identified inhibitor (p16) of cyclin-dependent kinase 4. MTS1 was homozygously deleted at high frequency in cell lines derived from tumors of lung, breast, brain, bone, skin, bladder, kidney, ovary, and lymphocyte. Melanoma cell lines that carried at least one copy of MTS1 frequently carried nonsense, missense, or frameshift mutations in the gene. These findings suggest that MTS1 mutations are involved in tumor formation in a wide range of tissues.

Base Sequence↗

PubMed1994

On the quantitative relationship between O6-methylguanine residues in genomic DNA and production of sister-chromatid exchanges, mutations and lethal events in a Mer- human tumor cell line.

O6-Methylguanine (m6G) is an altered base produced in DNA by SN1 methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This lesion is repaired by the protein O6-methylguanine-DNA methyltransferase (MGMT) in normal human cell lines, but is not repaired in certain human tumor lines that are termed Mex- or Mer-. Compared with repair-proficient cell lines, such repair-deficient tumor lines are hypersensitive to the production by MNNG of sister-chromatid exchanges (SCE), mutations and lethality. We report here that MNNG treatment produces 1 SCE for every 42 +/- 10 m6G formed in the genome of Mer- tumor cells, 1 6TG-resistant mutant for every 8 (range of 5-14) m6G produced statistically in the coding region of the hypoxanthine phosphoribosyltransferase gene, and 1 lethal event per 6650 +/- 1200 m6G. In addition, in vitro base mismatch incision at m6G: BrU pairs was similar to that at m6G: T pairs, the lesions that likely initiate SCE production. We conclude that m6G residues in genomic DNA are very recombinogenic as well as highly mutagenic in Mer- human tumor cells. The results are interpreted in terms of the relationship between methylation-induced SCE and G: T mismatch recognition.

Base Sequence↗

PubMed1993

Localization of chromosome 9p homozygous deletions in glioma cell lines with markers constituting a continuous linkage group.

Southern blot analyses of the 9p-localized type I interferon (IFN) genes in DNAs obtained from malignant glioma cell lines and glial tumor tissue have indicated that homozygous deletions of the IFN-alpha and IFN-beta genes often occur during the development of the highly malignant central nervous system neoplasm, glioblastoma. We have applied a set of markers that span the IFN region on 9p to the analysis of DNAs from 30 human glioma cell lines in order to define the region of homozygous deletion associated with this cancer more precisely. Fourteen of the cell lines revealed either complete (12 cases) or partial (2 cases) homozygous deletions of the IFN-alpha gene cluster; no instances of homozygous deletions were observed that did not involve the IFN-alpha region. Genomic DNA identified by the markers nearest to and flanking the IFN-alpha genes were retained in 5 of the cases with homozygous deletions. Consequently, these results limit the extent of homozygous deletions in glioma cell lines to a small region of 9p21-p22 that includes most of the type I IFN locus.

Base Sequence↗

PubMed1993

DNA-substrate sequence specificity of human G:T mismatch repair activity.

G:T mispairs in DNA originate spontaneously via deamination of 5-methylcytosine. Such mispairs are restored to normal G:C pairs by both E. coli K strains and human cells. In this study we have analyzed the repair by human cell extracts of G:T mismatches in various DNA contexts. We performed two sets of experiments. In the first, repair was sequence specific in that G:T mispairs at CpG sites at four different CpG sites were repaired, but a G:T mismatch at a GpG site was not. Cytosine hemimethylation did not block repair of a substrate containing a CpG/GpT mismatch. In the second set of experiments, substrates with a G:T mismatch at a fixed position were constructed with an A, T, G, or C 5' to the mismatched G, and alterations in the complementary strand to allow otherwise perfect Watson-Crick pairing. All were incised just 5' to the mismatched T and competed for repair incision with a G:T substrate in which a C was 5' to the mismatched G. Thus human G:T mismatch activity shows sequence specificity, incising G:T mismatched pairs at some DNA sites, but not at others. At an incisable site, however, incision is little influenced by the base 5' to the mismatched G.

Base Composition↗

PubMed1993

Amplification of the gene encoding the alpha-subunit of the mitochondrial ATP synthase complex in a human retinoblastoma cell line.

A cDNA clone encoding the precursor of the alpha-subunit of the human mitochondrial ATP synthase (F1-ATPS) complex was isolated from a library prepared from the poly(A)+ RNA present in a retinoblastoma (RB) cell line. Northern blot analysis of RNAs derived from a variety of transformed cell lines as well as from normal human fetal tissues indicated that RNA expression was significantly higher in two of the four RB cell lines analysed, Y79 (10- to 30-fold) and RB522A (3- to 8-fold), than in other cell lines or tissues. The increased mRNA level was apparently the result of gene amplification in Y79, but not in RB522A.

Amino Acid Sequence↗

PubMed1993

Deoxyguanosine reverses inhibition by hydroxyurea of repair of UV-irradiated adenovirus 5.

Hydroxyurea, an inhibitor of ribonucleotide reductase, blocks the repair by normal human fibroblasts of ultraviolet light-damaged adenovirus 5. A mixture of all four DNA deoxynucleosides present with hydroxyurea reversed the inhibition, an effect consistent with the target of hydroxyurea being ribonucleotide reductase. Single deoxynucleosides were differentially effective in reversing the inhibition: deoxyguanosine reversed 100% of the HU block, deoxyadenosine 70%, deoxythymidine 55%, but deoxycytidine only 3%. The results are interpreted in the context of the known pattern of stimulation and inhibition of mammalian ribonucleotide reductase by dNTP effectors. We suggest that biological data may be important to assessing the availability of substrate for the ribonucleotide reductase molecules that provide dNTPs for DNA repair.

Adenoviridae↗

PubMed1993

Isolation of two cell lines from a human malignant glioma specimen differing in sensitivity to radiation and chemotherapeutic drugs.

Two aneuploid cell lines which differ in their inherent sensitivity to ionizing radiation and chemotherapeutic agents were established concurrently from a single tumor specimen obtained from a patient with glioblastoma. M059J cells are approximately 30-fold more sensitive to radiation than are M059K cells (surviving fractions at 2 Gy were 0.02 and 0.64, respectively). This relative difference in radiation sensitivity has remained a stable feature of the cell lines during 2 years in continuous culture. In addition, cells of the M059J line are more sensitive than those of the M059K line to the cytotoxic effects of bleomycin, N,N-bis(2-chloroethyl)-N-nitrosourea, and nitrogen mustard. These cell lines may prove to provide a useful model system for evaluating the cellular and molecular processes which confer resistance or sensitivity in cancer treatment.

Adult↗

PubMed1992

Incision at O6-methylguanine:thymine mispairs in DNA by extracts of human cells.

Human cell-free extracts were used to detect activities specifically incising O6-methylguanine (m6G) paired with C or T in DNA. A 45-bp double-stranded DNA containing one m6G across from a T (m6G:T) was the test substrate. Extracts from glioblastoma cell lines A172 and A1235 (lacking the m6G-specific repair protein m6G-DNA methyltransferase, MGMT) and colon carcinoma cell line HT29, containing MGMT, showed incision activities specific for the T strand of m6G:T [and G:T, as reported previously by Wiebauer and Jiricny (1989)] substrates, but did not cleave m6G:C (or G:C) substrates. Competition experiments showed that the activity was similar to, if not identical with, the activity in human cells that incises G:T mismatches. The incision sites were similar to those recognized by human G:T- or G:A-specific mismatch enzymes, i.e., the phosphodiester bonds both 3' and 5' to the poorly matched T, suggesting the glycolytic removal of the poorly matched T followed by backbone incisions by class I or II AP endonucleases. Three experiments in which MGMT was inactivated showed that the m6G:T incision activity was not simply due to a two-step mechanisms in which MGMT would first mediate conversion of the m6G:T substrate to a G:T substrate which would serve as a substrate for G:T incision. Extracts from HT29 contained a DNA-binding factor, possibly DNA sequence-specific, that inhibited incision of the m6G:T (but not the G:T) substrate, that was removed by the addition of synthetic DNA to the reaction.

Base Composition↗

PubMed1992

Heterogeneity in response to treatment with buthionine sulfoximine or interferon in human malignant glioma cells.

Two tumor cell lines were established from each of three human malignant glioma biopsy specimens (M059, M067, M071) and sensitivity to treatment with radiation or chemotherapeutic agents (BCNU, nitrogen mustard) was determined. The effects of recombinant human interferon-alpha (rIFN) on the radiation response and of buthionine sulfoximine (BSO) on the drug response were investigated as well. For tumor M059, two cell lines that differed significantly in radiosensitivity were isolated (surviving fractions at 2 Gy = 0.02 and 0.64). The chemosensitivity and response to chemical modification differed as well. Cell lines established from tumor M071 differed in their response to rIFN only and were not sensitized by BSO. M067 cell lines showed little difference and were not sensitized by either agent. These results suggest that differences may exist both within and among human malignant gliomas with regard to their sensitivity to drugs, radiation, and the ability of chemical agents to modify treatment responses.

Buthionine Sulfoximine↗

PubMed1992

Radiosensitivity testing of human primary brain tumor specimens.

The inherent radiosensitivity of early passage cells derived from 22 patients with tumors of glial origin has been determined using a clonogenic assay system. The mean (+/- SD) surviving fraction at 2 Gy was 0.37 +/- 0.22 (range = 0.02-0.87). No correlation between inherent radiosensitivity and tumor cell plating efficiency or intracellular glutathione was observed. Tumor cells that were both resistant to nitrosoureas and expressed the Mer+ phenotype did not differ significantly in their radiosensitivity as compared to cells that were repair deficient (Mer-) and sensitive to nitrosoureas. Initial clinical follow-up suggests that factors in addition to inherent tumor cell radiosensitivity, such as performance status and age, continue to be the most important determinants of the response of patients with primary brain tumors to radiotherapy.

Astrocytoma↗

PubMed1992

The selective use of AMSA following high-dose cytarabine in patients with acute myeloid leukaemia in relapse: a Leukemia Intergroup study.

This clinical trial was designed to evaluate the role of high-dose cytarabine (ara-C) in the treatment of adults with acute myeloid leukaemia (AML) in first relapse. We also tested the hypothesis that the selective use of AMSA (100 mg/m2/d on days 7, 8 and 9) would increase the complete remission (CR) rate when leukaemia cells remained in the bone marrow immediately following 6 d of Ara-C (2-3 g/m2/12 h) alone. Of 155 patients evaluable for response, 115 (74%) experienced marked cytoreduction by day 6 and received no further induction chemotherapy; 53 (45%) of these patients achieved CR after one course and 45 (38%) had resistant disease. The 36 patients (23%) with inadequate cytoreduction after the 6 d of ara-C alone were randomly assigned either to no further chemotherapy (21 patients) or to 3 d of AMSA (15 patients). The CR rates after one course were 14% and 53%, respectively (P = 0.01), and the fractions with resistant disease were 76% and 40%, respectively. The fractional reduction of leukaemia cells in the day 6 bone marrow aspirate specimen (P < 0.0001) and the reduction in the leukaemia cell mass measured in the day 6 marrow biopsy (P = 0.001) were the strongest predictors for achieving CR versus having residual disease in univariate analyses. The median duration of remission was 5 months, but seven patients (10%) remain in CR after 30-92 + months. Among the 140 patients who received only the 6 d of ara-C, the pretreatment albumin (P = 0.002) and lactate dehydrogenase (P = 0.01) levels were the strongest predictors of response in univariate analyses, but only the albumin remained significant (P = 0.01) in a stepwise logistic regression analysis. Those patients with albumin > 4.0 mg/dl and LDH < 125% of normal had a 71% CR rate, and only 16% had resistant disease. Thus, pretreatment characteristics and rapid cytoreductin in the day 6 bone marrow sample identified a favourable subset of patients with AML in first relapse, some of whom responded quite well to 6 d of ara-C alone and have had long disease-free remissions.

Acute Disease↗

PubMed1992

Lack of expression of tumor-suppressor genes in human malignant glioma cell lines.

Human malignant gliomas (glioblastomas and anaplastic astrocytomas) are the most frequent brain tumors and are associated with a variety of genetic alterations including retinoblastoma (RB) and p53 gene mutations, loss of interferon alpha and beta (IFNA, IFNB) genes and lack of O6-methylguanine-DNA methyltransferase (MGMT) expression. Yet, in the studies performed to date, the relationship between these alterations has not been addressed. In this report, we have studied gene expression in 29 malignant glioma cell lines and have determined that, although loss of the interferon genes and loss of RB, p53 and MGMT mRNAs are frequent events, combinations of genetic alterations involving these four proven or putative tumor-suppressor genes are relatively infrequent. The exception was loss of RB mRNA, which may be associated with lack of MGMT mRNA.

Chromosome Deletion↗

PubMed1991

Glutathione levels and chemosensitizing effects of buthionine sulfoximine in human malignant glioma cells.

Biopsy samples and cultured cells derived from them were obtained from 39 patients with malignant glioma and were analyzed for 1) glutathione (GSH) content; 2) sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and/or nitrogen mustard (HN2) treatment and 3) the effect of buthionine sulfoximine (BSO) treatment on BCNU and/or HN2 cytotoxicity. The average GSH concentration of biopsy specimens was lower than those of cultured cells (2.36 +/- 0.44 vs. 11.42 +/- 2.32 nmol/10(6) cells). While some of the tumor specimens were sensitive to either BCNU or HN2, the majority were resistant to both. However, 8 of 23 tumors tested showed enhanced sensitivity to BCNU following treatment with BSO. Five of 17 tumors were similarly sensitized to HN2 by BSO. These results suggest that BSO chemosensitization may be of value for certain patients and that screening assays may help identify treatment-sensitive individuals.

Adolescent↗