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Biomedical subjects

J P Th'ng

Publications and source records attributed to J P Th'ng.

17 recordsLinked to original sources
PubMed2001

Expression of p21Waf1/Cip1 and cyclin D1 is increased in butyrate-resistant HeLa cells.

Sodium butyrate induced cell cycle arrest in mammalian cells through an increase in p21Waf1/Cip1, although another study showed that this arrest is related to pRB signaling. We isolated variants of HeLa cells adapted to growth in 5 mm butyrate. One of these variants, clone 5.1, constitutively expressed elevated levels of p21Waf1/Cip1 when incubated in regular growth medium and in the presence of butyrate. Despite this elevated level of p21Waf1/Cip1, the cells continue to proliferate, albeit at a slower rate than parental HeLa cells. Western blot analyses showed that other cell cycle regulatory proteins were not up-regulated to compensate for the elevated expression of p21Waf1/Cip1. However, cyclin D1 was down-regulated by butyrate in HeLa cells but not in clone 5.1. We conclude that continued expression of cyclin D1 allowed clone 5.1 to grow in the presence of butyrate and elevated levels of p21Waf1/Cip1.

Blotting, Western↗
PubMed2001

Histone modifications and apoptosis: cause or consequence?

Since the first description of apoptosis, genetic and biochemical studies have led to a greater understanding of the multiple pathways that eukaryotic cells can take to terminate their existence. These findings have also proven useful in understanding the development of various diseases such as AIDS, Alzheimer's, and Parkinson's and have provided potential targets for possible therapies. Despite all these studies, the mechanism of chromatin condensation, a morphological hallmark of apoptosis, remains elusive. This review describes the work to date on the post-translational modifications of histones during apoptosis and discusses the models that have been presented to explain the apoptotic condensation of chromatin.

Acetylation↗
PubMed2000

Rapid exchange of histone H1.1 on chromatin in living human cells.

The considerable length of DNA in eukaryotic genomes requires packaging into chromatin to fit inside the small dimensions of the cell nucleus. Histone H1 functions in the compaction of chromatin into higher order structures derived from the repeating 'beads on a string' nucleosome polymer. Modulation of H1 binding activity is thought to be an important step in the potentiation/depotentiation of chromatin structure for transcription. It is generally accepted that H1 binds less tightly than other histones to DNA in chromatin and can readily exchange in living cells. Fusion proteins of Histone H1 and green fluorescent protein (GFP) have been shown to associate with chromatin in an apparently identical fashion to native histone H1. This provides a means by which to study histone H1-chromatin interactions in living cells. Here we have used human cells with a stably integrated H1.1-GFP fusion protein to monitor histone H1 movement directly by fluorescence recovery after photobleaching in living cells. We find that exchange is rapid in both condensed and decondensed chromatin, occurs throughout the cell cycle, and does not require fibre-fibre interactions. Treatment with drugs that alter protein phosphorylation significantly reduces exchange rates. Our results show that histone H1 exchange in vivo is rapid, occurs through a soluble intermediate, and is modulated by the phosphorylation of a protein or proteins as yet to be determined.

Chromatin↗
PubMed1998

Chromatin condensation is not associated with apoptosis.

Apoptosis plays an important role in the survival of an organism, and substantial work has been done to understand the signaling pathways that regulate this process. Characteristic changes in chromatin organization accompany apoptosis and are routinely used as markers for cell death. We have examined the organization of chromatin in apoptotic PC12 and HeLa cells by indirect immunofluorescence and electron spectroscopic imaging. Our results indicate that de novo chromatin condensation normally seen during mitosis does not occur when cells undergo apoptosis. Instead, the condensed chromatin typically observed results from aggregation of the heterochromatin. We present evidence that, early in apoptosis, there is a rapid degradation of the nuclease-hypersensitive euchromatin that contains hyperacetylated histones. This occurs coincident with the loss of nuclear integrity due to degradation of lamins and reorganization of intranuclear protein matrix. These events lead to collapse of the nucleus and aggregation of heterochromatin to produce the appearance of condensed apoptotic chromatin. This heterochromatin aggregate is then digested by nucleases to produce the oligonucleosomal DNA ladder that is a hallmark of late apoptosis. Unlike mitosis, we have not seen any evidence for the requirement of phosphorylated histones H1 and H3 to maintain the chromatin in the condensed state.

Animals↗
PubMed1997

Four distinct cyclin-dependent kinases phosphorylate histone H1 at all of its growth-related phosphorylation sites.

In mammalian cells, up to six serines and threonines in histone H1 are phosphorylated in vivo in a cell cycle dependent manner that has long been linked with chromatin condensation. Growth-associated H1 kinases, now known as cyclin-dependent kinases (CDKs), are thought to be the enzymes responsible for this process. This paper describes the phosphorylation of histone H1 by four different purified CDKs. The four CDKs phosphorylate only the cell cycle specific phosphorylation sites of H1, indicating that they belong to the kinase class responsible for growth-related H1 phosphorylation in vivo. All four CDKs phosphorylate all of the interphase and mitotic-specific H1 sites. In addition to the (S/T)PXK consensus phosphorylation sites, these four CDKs also phosphorylate a mitotic-specific in vivo H1 phosphorylation site that lacks this sequence. There is no site selectivity among the growth-related phosphorylation sites by any of the four CDKs because all four CDKs phosphorylate all relevant sites. The results imply that the cell cycle dependent H1 phosphorylations observed in vivo must involve differential accessibility of H1 sites at different stages of the cell cycle.

Animals↗
PubMed1996

A brief staurosporine treatment of mitotic cells triggers premature exit from mitosis and polyploid cell formation.

At any point during the progression of many tumor types, cells can develop a hyperploid DNA content. Hyperploid tumors are significant more aggressive, with a higher growth rate and a poor patient prognosis. Yeast genetics have implicated three important genes involved in DNA ploidy changes: cdc2, cyclin b, and a specific inhibitor of the p34(cdc2)/cyclin B kinase, rum1. Mutations in these genes uncoupled the dependence mitosis on DNA replication in the fission yeast, Saccharomyces pombe. It was proposed that the inactivation of the mitotic kinase complex, p34(cdc2)/cyclin B, induces a G(1), state wherein the cells re-replicate their DNA without an intervening mitosis. We show in this report that treatment of only M phase-arrested mouse cells, with the protein kinase inhibitor staurosporine, induced polyploidy. Nocodazole-arrested metaphase FT210 cells were pulsed with 100 ng/ml of staurosporine for 1 h. This 1-h treatment results in the inhibition of the mitotic p34(cdc2) kinase. The inhibition of the mitotic kinases leads to a reduction in the histone H1 and H3 mitotic-associated phosphorylations, chromosome decondensation and nuclear membrane reformation. When released into normal growth medium, these cells are reset to a G(1)state, re-replicate their DNA without completing mitosis, and become octaploid.

Alkaloids↗
PubMed1996

A ribonucleotide reductase inhibitor, MDL 101,731, induces apoptosis and elevates TRPM-2 mRNA levels in human prostate tumor xenografts.

MDL 101,731, (E)2'-fluoromethylene-2'-deoxycytidine, is an irreversible inhibitor of ribonucleotide diphosphate reductase and causes regression of human tumors in nude mouse models. Messenger RNA levels for testosterone-repressed prostatic message-2 (TRPM-2), a transcript that increases in human tumor xenografts undergoing programmed cell death, were analyzed by in situ hybridization. Xenografts derived from a human prostate tumor cell line (PC-3) regressed following treatment with MDL 101,731 and the relative levels of TRPM-2 mRNA increased up to threefold in drug-treated animals. Apoptosis in the tumor xenografts was further indicated by in situ labeling of DNA strand breaks by incorporation of biotinylated-dUTP with terminal deoxynucleotidyl transferase. In vitro, PC-3 cells incubated with MDL 101,731 showed evidence of apoptosis based on flow cytometry and DNA laddering. These data support the hypothesis that MDL 101,731 stimulates programmed cell death in regressing PC-3 xenografts.

Animals↗
PubMed1995

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.

Alkaloids↗
PubMed1994

Inhibition of histone phosphorylation by staurosporine leads to chromosome decondensation.

In mammalian cells, hyperphosphorylation of histone H1 and phosphorylation of histone H3 correlate well with the G2 phase to metaphase condensation of chromosomes, and these phosphorylations most probably have a role in initiating and controlling the entry into mitosis. The protein kinase inhibitor staurosporine has been used to examine the role of H1 and H3 phosphorylations in controlling chromosome condensation in the mouse FM3A cell line. We present evidence that (i) staurosporine inhibits the protein kinases that phosphorylate histone H1 during mitosis, (ii) staurosporine also inhibits the histone H3-specific kinase, (iii) the inhibition of these kinase activities prevent cells from entering mitosis, and (iv) addition of staurosporine to cells already arrested at metaphase by nocodazole causes a rapid dephosphorylation of histones H1 and H3 and the decondensation of the metaphase chromosomes. The results show that the hyperphosphorylation of histone H1 and phosphorylation of histone H3 are required to maintain metaphase chromosomes in their condensed state.

Alkaloids↗
PubMed1992

Intrinsically bent DNA flanks both sides of an RNA polymerase I transcription start site. Both regions display novel electrophoretic mobility.

We have identified two intrinsically bent regions of DNA which flank the transcription start site of the rRNA gene from Physarum polycephalum. DNA fragments from both regions were analyzed by circular permutation polyacrylamide gel electrophoresis assay and computer modeling. Both types of analysis indicate that one fragment contains a relatively simple bend centered about 160 base pairs (bp) upstream of the transcription start site while the other fragment contains multiple bends, the most prominent of which is centered about 150 bp downstream of the start site. According to both gel mobilities and computer modeling we estimate that the net bending in each is about 45 degrees. These fragments were studied in detail by varying parameters of electrophoresis that are known to affect bending. Previous work indicates that anomalous mobility should decrease when temperature or ethidium bromide concentration is increased, whereas anomalous mobility should increase when polyacrylamide gel percentage is increased. The anomalous mobility of both fragments decreases as temperature is raised from 4 to 65 degrees C, although the bent structure centered at -160 bp is more temperature labile than the bend at +150 bp. Strikingly different behavior was observed for the two fragments as the polyacrylamide concentrations was varied. As polyacrylamide concentrations are increased from 6 to 10%, the anomalous mobility of the bend centered at -160 bp increases while that of the bend centered at +150 bp decreases. The bend centered at +150 bp is "straightened" at all ethidium concentrations tested. In sharp contrast and unexpectedly, the anomalous migration of the bend centered at -160 bp increases dramatically in 0.1 micrograms/ml ethidium bromide. Many of the mobility differences we observe suggest that the two regions studied represent structurally distinct forms of bent DNA. The location of these strongly bent regions on either side of a RNA polymerase I transcription start site suggests important roles for such structures in chromatin structure and transcription initiation.

Animals↗
PubMed1991

Molecular cloning of a rat testis form of the inhibitor protein of cAMP-dependent protein kinase.

The form of inhibitor protein of the cAMP-dependent protein kinase (PKI) that has been most thoroughly studied is a protein purified from rabbit skeletal muscle. Beale et al. previously isolated a species of PKI from rat testis that appeared from its amino acid composition to be quite distinct from the rabbit skeletal muscle protein [Beale, E. G., Dedman, J. R. & Means, A. R. (1977) J. Biol. Chem. 252, 6322-6327]. The amino acid sequence of a form of rat testis PKI has now been determined both by sequencing overlapping peptide fragments for 95% of the protein and by the isolation of a cDNA clone containing the coding region for the 70-amino acid protein. The sequence of the 70-amino acid testis PKI displays a maximum of only 41% sequence identity with the previously sequenced 75-amino acid rabbit skeletal muscle PKI. However, the two forms have identical potency as inhibitors and the key amino acids of the pseudosubstrate site, shown to be critical for maximal inhibition with the rabbit skeletal muscle PKI, have been conserved in the testis protein. The rabbit skeletal muscle and rat testis PKIs most likely represent distinct isoforms. The nucleotide sequence of the rat testis PKI cDNA suggests that a second form of testis PKI, longer by 8 additional amino-terminal amino acids, might also be produced.

Amino Acid Sequence↗
PubMed1990

The FT210 cell line is a mouse G2 phase mutant with a temperature-sensitive CDC2 gene product.

The mouse cell FT210 was isolated as a G2 phase mutant with a possible defect in the histone H1 kinase. We determined that a temperature-sensitive lesion in this cell line lies in the CDC2 gene. DNA sequence analysis revealed two point mutations in highly conserved regions of the gene: an isoleucine to valine change in the PSTAIR region, and a proline to serine change at the C-terminal region of the protein p34. These mutations cause the p34 protein kinase to become inactivated and degraded in FT210 cells at the restrictive temperature, 39 degrees C. The consequence of this temperature-induced inactivation of the CDC2 gene product is cell cycle arrest at the mid to late G2 phase, and this arrest can be alleviated by the introduction of the human CDC2 homolog.

Amino Acid Sequence↗
PubMed1986

Excision repair of DNA in the presence of aphidicolin.

During excision repair of UV light or dimethyl sulphate (DMS)-induced damage to DNA the patch size for actively replicating KB or T98G cells is around 20 nucleotides. When confluent T98G cells or 'quiescent' KB cells are used the patch size is around 10 nucleotides. This value can be increased to around 20 nucleotides in T98G cells if a large excess of BrdUrd is included in the repair incubation medium. With 'quiescent' KB cells the patch size is not increased by excess BrdUrd. For all of these experimental conditions, when excision repair of UV or DMS damage takes place in the presence of aphidicolin, the patch size is found to be several times that found in its absence. Given the inhibitory specificity of aphidicolin for DNA polymerase alpha these results provide additional evidence that DNA polymerase alpha plays a role in the excision repair of DNA damaged by UV light or DMS. It is postulated that aphidicolin interrupts the processivity of the DNA polymerase alpha holoenzyme and allows an exonuclease to enlarge the repair site.

Aphidicolin↗
PubMed1984

3-Aminobenzamide does not increase repair patch size in mammalian cells.

Several groups of investigators have reported that the extent of repair replication following treatment of mammalian cells with dimethyl sulfate is increased by 3-aminobenzamide (3AB) over that occurring in its absence. Two plausable explanations for this phenomenon were tested. The first is that the number of nucleotides inserted per repair site, that is the repair patch size, is increased and the second is that the rate of repair is increased. Human T98G cells were treated with dimethyl sulfate and allowed to repair their DNA in the presence or absence of 3AB. It was found that the presence of 3AB did not increase the repair patch size nor the rate of removal of methylation products from the DNA, which is assumed to equal the rate of repair. The results of further experiments suggested that the 3AB effect can be explained by changes in nucleotide precursor pools.

Benzamides↗
PubMed1983

DNA repair patch size measurements with nucleosomal DNA.

A modified method for measuring DNA repair patch sizes by the bromodeoxyuridine buoyant density shift method is presented. This method involves the digestion of total chromatin by Staphylococcal nuclease to produce small DNA fragments with a very homogeneous size distribution. Using this method to measure the size of repair patches following treatment with u.v.-light or dimethyl sulfate, a calculated incorporation of 40 nucleotides was obtained. This value is identical to previously published patch size estimations obtained by the buoyant density shift method with sonically produced DNA fragments. It is also in close agreement with values recently obtained by the bromodeoxyuridine photolysis method. Taken together, these independent results obtained with a variety of DNA damaging agents suggest that excision repair patch size is independent of the damaging agent.

Bromodeoxyuridine↗
PubMed1982

Excision-repair patch size in DNA from human KB cells treated with UV-light, or methyl methanesulfonate.

We have used the method of combined bromodeoxyuridine density label and radioactive label to measure the size of the repair patches appearing in the DNA of KB cells following treatment with UV-light or MMS. The repair patch size distribution was found to be the same for both agents, corresponding to the insertion of 34-40 nucleotides. These results, confirm recent results obtained by the bromodeoxyuridine-photolysis technique, that simple alkylating agents induce 'long patch' repair in human cells.

Bromodeoxyuridine↗