Biomedical subjects

R S Day

Publications and source records attributed to R S Day.

At least 19 recordsLinked to original sources

PubMed1998

Correlation of B-FABP and GFAP expression in malignant glioma.

The murine brain fatty acid binding protein (B-FABP) is encoded by a developmentally regulated gene that is expressed in radial glial cells and immature astrocytes. We have cloned the human B-FABP gene and have mapped it to chromosome 6q22-23. We show that B-FABP mRNA is expressed in human malignant glioma tumor biopsies and in a subset of malignant glioma cell lines, as well as in human fetal retina and brain. Malignant glioma tumors are characterized by cytoplasmic bundles of glial fibrillary acidic protein (GFAP), a protein normally expressed in mature astrocytes. Establishment of malignant glioma cell lines often results in loss of GFAP. The subset of malignant glioma cell lines that express GFAP mRNA also express B-FABP mRNA. Co-localization experiments in cell lines indicate that the same cells produce both GFAP and B-FABP. We suggest that some malignant gliomas may be derived from astrocytic precursor cells which can express proteins that are normally produced at different developmental stages in the astrocytic differentiation pathway.

Amino Acid Sequence↗

PubMed1998

The integration of a novice user interface into a professional modeling tool.

This paper describes a software tool, the Oncology Thinking Cap (OncoTCAP) and reports on our efforts to develop a novice user interface to simplify the task of describing biological models of cancer and its treatment. Oncology Thinking Cap includes a modeling tool for making relationships explicit and provide dynamic feedback about the interaction between cancer cell kinetics, treatments, and patient outcomes. OncoTCAP supports student learning by making normally invisible processes visible and providing a representational tool that can be used to conduct thought experiments. We also describe our novice interface and report the results of initial usability testing.

Cell Cycle↗

PubMed1997

Vitamin D inhibition of prostate adenocarcinoma growth and metastasis in the Dunning rat prostate model system.

OBJECTIVES: Risk factors for prostate cancer (PCa)-related mortality include old age, black race, and residence in northern latitudes. The objectives of this study are to examine the in vitro and in vivo effects of 1,25-dihydroxycholecalciferol (1,25-D3) and less-hypercalcemic analogues on the Dunning rat prostate adenocarcinoma model. METHODS: To evaluate the effect of 1,25-D3 on PCa in vitro, we used the highly metastatic Mat-lylu (MLL) and moderately metastatic R3327-AT-2 (AT-2) Dunning prostate cell lines, and examined effects on growth, clonogenicity, differentiation, and cell cycle. In vivo analysis included examination of the effects of these compounds on tumor growth and metastasis. RESULTS: Using both the 3-day MTT and 7-day clonogenic assay, 1,25-D3 demonstrated a growth inhibitory effect with a concentration for 50% inhibition (IC50) of approximately 20 microM for both MLL and AT-2. Cell cycle analysis of treated MLL cells (10 microM 1,25-D3 for 48 hours) had 25% more cells in the G0/G1 phase than did control cells. To examine the in vivo effect of 1,25-D3 and the less hypercalcemic vitamin D analogue, Ro25-6760 (or 6760), on MLL PCa growth and metastasis, tumors (5 x 10(5) cells) were implanted subcutaneously into the flank of Copenhagen rats on the same day that treatment was initiated with 1,25-D3 (1 microgram) or 6760 (1 or 5 micrograms); rats received treatment three times a week. After 3 weeks, 1,25-D3 and 6760 (5 micrograms dosing) resulted in an inhibition of tumor volume and a reduction in the number and size of lung metastases. CONCLUSIONS: These preclinical studies demonstrate the profound in vitro, or in vivo, or both antiproliferative and differentiating effects of 1,25-D3 and 6760 on PCa and suggest that these drugs may have potential beneficial effects in the treatment of advanced PCa.

Adenocarcinoma↗

PubMed1997

Increased radiosensitivity of p16 gene-deleted human glioma cells after transfection with wild-type p16 gene.

The A1235 and T98 cell lines derived from human gliomas have homozygous deletions in their p16 genes and are radiosensitive and radioresistant, respectively, with respect to other established glioma cell lines. These differences in radiosensitivity may be due to variations to some extent among cell lines, rather than genetically defined resistance or sensitivity. We examined the effect on radiation sensitivity of introducing a wild-type p16 gene into both p16-deficient glioma cell lines. The plasmid pOPMTS containing human wild-type p16 cDNA and a neomycin resistance gene, or the control plasmid pOPRSV1, were transfected into these cells. Clones from both cell lines, which expressed wild-type p16 mRNA constitutively after transfection with pOPMTS, were more radiosensitive than the parental cells and clones obtained after transfection with the negative control plasmid.

Carrier Proteins↗

PubMed1997

Intact G2-phase checkpoint in cells of a human cell line lacking DNA-dependent protein kinase activity.

Cells respond to radiation-induced DNA damage in a cell cycle phase-specific manner as shown by (1) variation in radiosensitivity across the cell cycle and (2) checkpoints in G1 and G2 phase at which arrest of progression of cells through the phases of the cell cycle occurs. We studied these processes in cells of human glioma cell lines which lack (M059J(PK-)) or express (M059K(PK+)) DNA-dependent protein kinase (DNA-PK) activity. Cell populations enriched with cells of a specific cell cycle phase were y-irradiated and analyzed for cell survival. Although both cell lines were relatively sensitive in G1 phase and resistant in S phase, the differential sensitivity was greater in M059J(PK-) cells. In the studies on checkpoints, unsynchronized cells were irradiated and examined for evidence of cell cycle arrest. Neither cell line showed a postirradiation G1-phase arrest, presumably because of mutant p53 status. For M059J(PK-) cells, all doses tested (2.5-10 Gy) resulted in a significant increase in the proportion of G2/M-phase cells; however, for M059K(PK+) cells, a significant increase in G2/M phase was observed only after 10 Gy. These results suggest that the ability to activate the G2-phase checkpoint remains intact in cells which lack DNA-PK activity.

Cell Cycle↗

PubMed1996

Base analog and neighboring base effects on substrate specificity of recombinant human G:T mismatch-specific thymine DNA-glycosylase.

We studied the substrate specificity of the human G:T mismatch-specific thymine glycosylase that initiates the repair of G:T and G:U base mismatches to G:C base pairs. Such mismatches arise when 5-methylcytosine or cytosine deaminate spontaneously (and hydrolytically) in DNA. Substrates were 45-bp DNA heteroduplexes that bore single G:T, m6G:T, 2,6-diaminopurine:T, 2-amino-6-(methylamino)-purine:T, 2-aminopurine:T, and G:m4T mispairs. The bases 5' to the poorly matched G were altered in selected G:T substrates to yield mispairs in four different contexts, ApG, CpG, GpG, and TpG. The recombinant thymine glycosylase was incubated with the 45-bp DNA substrates, each labeled at the 5'-terminus of the strand containing the mismatched T. The DNAs were then treated with 0.1 N NaOH to catalyze phosphodiester bond breakage at the newly-generated AP sites, and the products were analyzed on DNA sequencing gels. As indicated by the amounts of the 20-nt incision product, the removal of the thymine base by the enzyme increased linearly between 0 and 40 min at which time the generation of product from all substrates ceased, probably because of enzyme inactivation. The rate of incision was greatest (0.7 fmol/min) with DNA containing the G:T mispair followed by the DNA containing the m6G:T mispair (0.38 fmol/min) and the DNA with the 2-amino-6-(methylamino)purine:T mispair (0.15 fmol/ min); the extent of reaction was 90%, 40%, and 20% respectively. By contrast to previous findings with cell-free extracts, DNA substrates containing 2,6-diaminopurine:T, 2-aminopurine:T, and G:m4T mispairs were not incised (< 2%). The amount of incision of the 45-bp DNA substrates containing G:T mispairs in the CpG context was 3-12-fold greater than in the TpG, GpG, and ApG contexts.

5-Methylcytosine↗

PubMed1996

Replication of O6-methylguanine-containing DNA by repair and replicative DNA polymerases.

The biological consequences of O6-methylguanine (m6G) in DNA are well recognized. When template m6G is encountered by DNA polymerases, replication is hindered and trans-lesion replication results in the preferential incorporation of dTMP opposite template m6G. Thus, unrepaired m6G in DNA is both cytotoxic and mutagenic. Yet, cell lines tolerant to m6G in DNA have been isolated, which indicates that some cellular DNA polymerases may replicate m6G-containing DNA with reasonable efficiency. Previous reports suggested that mammalian pol beta could not replicate m6G-containing DNA, but we find that pol beta can catalyze trans-lesion replication; however, the lesion must reside in the optimal context for pol beta activity, single- or short nucleotide gapped substrates. Primed single-stranded DNA templates, with or without template m6G, were poor substrates for pol beta as reported in earlier studies. In contrast, trans-lesion replication by bacteriophage T4 DNA polymerase was observed for primed single-stranded DNA templates. Replication of m6G-containing DNA by T4 DNA polymerase required the gp45 accessory protein that clamps the polymerase to the DNA template. The rate-limiting step in replicating m6G-containing DNAs by both DNA polymerases tested was incorporation of dTMP across from the lesion.

Base Sequence↗

PubMed1995

Lack of correlation between DNA-dependent protein kinase activity and tumor cell radiosensitivity.

Lack of DNA-dependent protein kinase (DNA-PK) activity confers radiosensitivity and defective DNA double-strand break repair. Nine human malignant glioma cell lines were studied to determine whether differences in DNA-PK activity reflect differences in inherent radiosensitivity or are predictive of tumor treatment response. DNA-PK activity was present in all cell extracts, as were the DNA-PK proteins, DNA-PK catalytic subunit, Ku p70, and Ku p80. No correlation was found between the levels of DNA-PK activity and inherent radiosensitivity or in the tumor treatment response. These preliminary results suggest that variation in DNA-PK activity may not be a determinant of clinical response in malignant glioma.

DNA-Activated Protein Kinase↗

PubMed1995

Incision at diaminopurine: thymine base pairs but not at guanine:O4-methylthymine base pairs in DNA by extracts of human cells.

Cell-free extract from the A1235 human malignant glioma cell line was employed to study the possibility of incision at 2,6-diaminopurine:T (DiAP:T), 2-amino-6-methylaminopurine:T (AMAP:T), and G:O4-methylthymine (G:m4T) mismatches, each placed in a 45 bp DNA at a defined site. The incision of a 45 bp DNA containing a G:T mispair at the same site was followed to determine the relationship between base pair structure and repair activity (ies) in the extract. The cell-free extract incised DNAs containing DiAP:T, AMAP:T, and G:T pairs similarly. Reminiscent of the known pattern of incision at G:T mismatches, products from each substrate were consistent with two incisions, one immediately 5' and one immediately 3' to the mismatched T, and only in the strand containing the mismatched T. While DNA with an O6-methylguanine:T (m6G:T) pair was also incised, DNA containing the G:m4T pair was not, but was rendered inciseable by pretreatment with O6-methylguanine-DNA methyltransferase. Incision of DiAP:T-containing DNA by the extract was less in the presence of unlabeled DNA containing G:T mispairs than in the presence of A:T- or G:A-containing DNA or in the absence of competing DNA. We suggest that the mechanism operating on DiAP:T and/or AMAP:T pairs may be the same as the human G:T repair pathway, possibly initiated by the action of a glycosylase as described by Wiebauer and Jiricny [Wiebauer, K., & Jiricny, J. (1989) Nature 339, 234-236; Wiebauer, K., & Jiricny, J. (1990) Proc. Natl. Acad. Sci, U.S.A. 87, 5842-5845].(ABSTRACT TRUNCATED AT 250 WORDS)

2-Aminopurine↗

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↗