Biomedical subjects
Publications and source records attributed to D L Slate.
Bioactivity-guided fractionation of the extracts of the green brittle star Ophiarachna incrassata using a protein tyrosine kinase pp60v-src inhibition assay led to the isolation of a new sterol sulfate [2] together with four known ones (1,3-5). All five compounds were found to inhibit protein tyrosine kinase pp 60v-src.
The marine polyketide natural product, halenaquinone, was shown to be an irreversible inhibitor of pp60v-src, the oncogenic protein tyrosine kinase encoded by the Rous sarcoma virus. This compound had an IC50 of approximately 1.5 microM against pp60v-src and also inhibited the ligand-stimulated kinase activity of the human epidermal growth factor receptor with an IC50 of approximately 19 microM. Halenaquinone blocked the proliferation of a number of cultured cell lines, including several transformed by oncogenic protein tyrosine kinases. Halenaquinol, xestoquinone, halenaquinol sulfate, and several simple synthetic quinone analogs were also shown to inhibit pp60v-src.
A series of doxorubicin-resistant variants of the human LS174T colon carcinoma cell line was generated by stepwise selection. These variants also exhibited increased resistance to vinblastine, etoposide, cis-platinum, and melphalan, suggesting that resistance was multifactorial. The parental LS174T cell line and 3 resistant variants were examined for over-expression of P-glycoprotein, changes in total cellular glutathione content, and the level of topoisomerase-II expression. Changes in all of these parameters were observed in the doxorubicin-selectants, along with a marked shift in the intracellular distribution of doxorubicin. P-glycoprotein RNA and protein levels were increased 2- to 3-fold in the resistant variants, while total glutathione levels increased 1.4- to 2.1-fold. Treatment with DL-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione biosynthesis, was able to reverse resistance to cis-platinum and melphalan in these variants, but had little effect on doxorubicin resistance. Immunoblot analysis of cell extracts indicated that the level of DNA topoisomerase II (EC 5.99.1.3) in the doxorubicin-resistant LS174T cells was decreased by approximately 50% compared with the parental cell line. Doxorubicin was mainly localized to the cytoplasm in resistant cells, while in the parent line it was mostly found in the nucleus. This constellation of changes suggests that selection with doxorubicin activated several mechanisms of resistance involving drug transport, metabolism, and ability to reach nuclear target sites.
Flow cytometry has been used to measure doxorubicin (DOX) retention in several pairs of drug-sensitive and multidrug-resistant (MDR) cell lines and in unselected human tumor cell lines. Co-exposure to several agents that have been reported to reverse multidrug resistance, particularly calcium entry blockers (CEBs), produced a dose-dependent increase in DOX accumulation in MDR cell lines. In MDR Chinese hamster ovary cells (CHRC5), DOX levels declined rapidly following removal of CEBs, reaching a plateau value above that found in cells treated with DOX alone; this small increase probably represents DOX that is not accessible to the p170 efflux pump overexpressed in these cells. Increased DOX retention could be observed even after brief exposure to CEBs and washout and correlates with a decrease in cell proliferation over a 3-day growth assay. These results suggest that only a brief inhibition of drug efflux is sufficient to produce a meaningful reversal of drug resistance.
A series of drug-resistant variants of the murine Lewis lung carcinoma (3LL-CK) cell line has been isolated using stepwise selection in increasing concentrations of doxorubicin. These variants exhibited classical multidrug resistance as evidenced by decreased doxorubicin accumulation, cross-resistance to vinblastine, reversibility of resistance by verapamil, and overexpression of P-glycoprotein. When the doxorubicin-resistant 3LL-CK cell populations were injected into the tail veins of B6D2F1 mice, their metastatic abilities were consistently reduced compared with that of the parental line.
A cDNA clone (pAE20-4) corresponding to the 1.3-kilobase human beta 2 interferon mRNA was used as a probe in blot-hybridization experiments of DNA from a panel of human-rodent somatic cell hybrids containing overlapping subsets of human chromosomes. The DNA hybridization experiments showed that the human beta 2 interferon gene is located on human chromosome 7. This assignment is consistent with previous experimental data in which the expression of the translationally active 1.3-kilobase beta 2 interferon mRNA was assayed in various somatic cell hybrids. Blot-hybridization experiments using DNA from different human cell strains and cell lines reveal distinct EcoRI restriction fragment length polymorphisms of the human beta 2 interferon gene.
A DNA subclone (pPE-4000) derived from the lambda B4 interferon-beta-related human genomic DNA clone was used as a probe in blot-hybridization experiments of DNA from a panel of human-rodent somatic cell hybrids containing overlapping subsets of human chromosomes. The DNA hybridization experiments showed that the lambda B4 IFN-beta locus is localized to human chromosome 4. A provisional regional assignment to 4q12-qter was also obtained. Thus available hybridization data implicate human chromosomes 2, 4, and 9 in the human IFN-beta system while the available biological data also implicated human chromosome 5.
Interferon-beta 1 (IFN-beta 1) complementary DNA was used as a hybridization probe to isolate human genomic DNA clones lambda B3 and lambda B4 from a human genomic DNA library. Blot-hybridization procedures and partial nucleotide sequencing revealed that lambda B3 is related to IFN-beta 1 (and more distantly to IFN-alpha 1). Analyses of DNA obtained from a panel of human-rodent somatic cell hybrids that were probed with DNA derived from lambda B3 showed that lambda B3 is on human chromosome 2. Similar experiments indicated that lambda B4 is not on human chromosomes 2, 5, or 9. The finding that DNA related to the IFN-beta 1 gene (and IFN-alpha 1 gene) is dispersed in the human genome raises new questions about the origins of the interferon genes.
Antisera raised against a human X mouse hybrid cell line containing human chromosome 21 as its only human chromosome, block induction of an antiviral state by human alpha interferon (IFN-alpha), block induction of (2'-5')oligoisoadenylate synthetase [2'-5')A synthetase), and block binding of 125I-labeled and 35S-labeled recombinant, human IFN-alpha A, but not 125I-labeled IFN-gamma, to cell surface receptors. The data presented clearly demonstrate that the cell surface receptors for IFN-alpha and IFN-gamma are different, and provide independent evidence of the role of a chromosome 21 coded cell surface molecule in the pathway to the generation of the antiviral state.
Analysis of human beta interferon (IFN) mRNA preparations obtained from poly(I) . poly (C)-induced human diploid fibroblasts (FS-4) and from several similarly induced human-mouse somatic cell hybrids by electrophoresis through agarose-CH3HgOH tube gels led to the detection of at least five translationally active human IFN-beta mRNA species. The results obtained are consistent with the existence of IFN-beta genes on different human chromosomes. Marked cell-dependent variability in the expression of these IFN mRNA species was observed.
To determine the chromosomal location of the human alpha interferon genes, we scored a series of human/rodent somatic cell hybrids for the presence of DNA sequences hybridizing to an alpha 1 interferon DNA probe. The presence of human chromosome 9 in a hybrid correlated with the presence of a family of alpha interferon genes.
The human fibroblast interferon gene beta 1 was mapped to human chromosome 9. Sequence homology with a beta 1 cDNA clone was detected in both genomic DNA and induced mRNA of human/mouse or human/hamster somatic cell hybrids containing human chromosome 9, but not in lines lacking this chromosome or those retaining a complex translocation involving chromosomes 9 and 11. Interferon mRNA that did not share sequence homology with the beta 1 cDNA clone was detected in lines containing human chromosomes 2 and 5 but lacking chromosome 9, suggesting the presence of other unlinked interferon sequences in the human genome.
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We investigated the cell cycle modulation of dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase, 7,8-dihydroxyfolate:NADP+ oxidoreductase, EC 1.5.1.3) levels in methotrexate-resistant Chinese hamster ovary cells synchronized by mitotic selection. DNA content and DHFR concentration were analyzed throughout the cell cycle by standard biochemical techniques and by double fluorescence staining utilizing the fluorescence-activated cell sorter. We found an S phase-specific period of DHFR biosynthetic activity. Commencing within hour 2 of S phase and continuing throughout the duration of S phase, there is a 90% increase in DHFR specific activity. This results from an approximately 2.5-fold increase in the level of DHFR, while total soluble protein increases 50% during the same period. This increase is the result of new synthesis of DHFR molecules initiated after the cell is physiologically committed to DNA replication. This increase in DHFR activity through S phage parallels the increasing rate of [3H]thymidine incorporation during the same interval. The maximum peak of DHFR activity is coincident with the maximum rate of DNA synthesis, both activities occurring during the bulk of DNA replication within the last stages of the 6.5-hr S phase.
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