Trichostatin A produces predominantly G 1 cell-cycle blockade and differentiation of the cisplatinum-sensitive A2780 ovarian cancer cell line. Given the propensity
of ovarian tumors to become ...resistant to cisplatinum, often leading to cross-resistance to other agents, we have extended
these observations by examining how the emergence of resistant phenotypes in A2780 cells affects the actions of histone deacetylase
(HDAC) inhibitors. Trichostatin A exposure (100 ng/mL, 24 hours) induced ultrastructural differentiation of the “intrinsically”
cisplatinum-resistant A2780-9M subline, with the reappearance of intercellular junctions and lumina containing primitive microvilli.
Similar trichostatin A exposure in the acquired resistance A2780CP cells produced minimal differentiation consisting of occasional
weak intercellular junctions. Independent of the differences in trichostatin A–induced differentiation, in both resistant
sublines trichostatin A produced a similar reduction in cell viability, by >90%, within 5 days of treatment. Diminished viability
in both A2780-9M and CP cells was associated with the absence of cell cycle arrest in G 1 , resulting in predominant G 2 -checkpoint arrest accompanied by a 10- to 20-fold increase in Annexin V binding and the reemergence of apoptosis. Similar
cell cycle arrests and apoptosis were also observed using other HDAC inhibitors and in other resistant ovarian cancer cell
lines (OVCAR-3 and SK-OV-3). Trichostatin A–induced apoptosis in resistant cells is in sharp contrast to its effects on the
parental cisplatinum-sensitive A2780 and normal MRC-5 fibroblast cell lines (predominant cycle arrest in G 1 with no detectable apoptosis). Western immunoblot analysis indicated trichostatin A triggers apoptosis in resistant ovarian
cancer cells via p53-independent activation of the intrinsic “mitochondrial” pathway, commensurate with induction of the Bcl-2–related
protein Bad. These results suggest cisplatinum resistance alters the effects of HDAC inhibition through a shift in cell cycle
arrest from the G 1 to the G 2 checkpoint and reactivation of the intrinsic mitochondrial apoptotic cascade.
Inhibitors of histone deacetylase activity are emerging as a potentially important new class of anticancer agents. In the current studies, exposing A2780 ovarian cancer cells to the histone ...deacetylase inhibitor trichostatin A (TSA) produced a marked change in cellular morphology, proliferation, and differentiation. Within 24 h of TSA treatment, there was a morphological transformation of the cells, with increased cytoplasm, a more epithelial-like columnar appearance, and the emergence of distinct cellular boundaries. Commensurate with the morphological transformation, TSA also inhibited cell proliferation; cells treated with TSA for 72 h increased to 110% of the initial cell numbers versus control cell numbers of 622%, with a corresponding reduction in mitotic activity and a flow cytometry S-phase fraction of 3.9% in TSA-treated cells versus 28.8% for control. TSA also induced epithelial-like differentiation with increased cytokeratin expression from 2% of controls to 22-25% of TSA-treated cells and the reappearance of intercellular plasma membrane junctions and primitive microvilli. Immunocytochemical analyses indicate the molecular mechanism underlying the actions of TSA on A2780 cell cycle progression and differentiation involves reexpression of the CDK inhibitor p21. Elevated levels of p21, in TSA-treated cells, were associated with a reduction in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). TSA also caused a decrease in the helix-loop-helix inhibitor of differentiation/DNA binding protein Id1, with no change in Id2 levels. In conclusion, the observed TSA-induced changes in p21, Rb, and Id1 are consistent with cell cycle senescence and differentiation of A2780 ovarian cancer cells.
Defects in the human MSH2 mismatch repair system have been implicated in cellular mutagenesis, tumorigenesis, and chemotherapeutic resistance. The current studies characterized the 5′ upstream ...proximal promoter region of thehMSH2 gene using transient transfection of A2780 ovarian cancer cells. Serial deletions of a 1.88-kb fragment of the proximal promoter region of the hMSH2 gene revealed that promoter activity was restricted to the first −281 bp. Targeted deletions within this −281 bp region coupled with specific sequence mutagenesis identified a response element for the p53 tumor suppressor protein located between −242 and −222 bp. The −242 hMSH2 p53 element is configured as a direct tandem repeat palindrome with 80% homology to the p53 consensus binding sequence. Co-transfection of anhMSH2 reporter and p53 expression vector into the p53-null cell line SK-OV-3 produced 10-fold enhanced transcription, which was lost when the −242 to −222 p53 binding site was mutated. These results clearly demonstrate the presence of a previously unidentified p53 response element in the hMSH2 proximal promoter. Its location at −242 bp upstream of the start site of transcription is distinct from two previously reported p53 sites at −447 and −416, which transactivate in Saos-2 cells (Scherer, S. J., Maier, S. M., Seifert, M., Hanselmann, R. G., Zang, K. D., Muller-Hermelink, H. K., Angel, P., Welter, C., and Schartl, M. (2000) J. Biol. Chem. 275, 37469–37473). Finally, in sharp contrast to their activity in Saos-2 cells, deletion of the −447 and −416 sites in A2780 cells had no effect onhMSH2 promoter activity. Thus, it appears that p53 regulates hMSH2 expression through multiple cell type-specific DNA response elements.
Immunohistochemical analysis confirmed the presence of MLH1 protein in A2780 ovarian cancer cells and its absence in this same cell line on acquired resistance to cisplatinum (A2780/CP). Transfection ...of a -1781-bp hMLH1 promoter construct into either A2780 or A2780/CP cells produced similar (30-fold) induction of luciferase, an indication that the transcriptional machinery for hMLH1 expression remains intact. hMLH1-luciferase activity was also unaffected by re-expression of hMLH1 following treatment of A2780/CP cells with the methylase inhibitor 2'-deoxy-5-azacytidine. Serial 5'-deletion studies of the hMLH1 promoter region in ovarian cancer cells localized transcriptional enhancers to a region (-250 to -151 bp) that excludes the previously identified CCAAT element (-282) active in HeLa cells. When these same deletion constructs were transfected into HeLa cells, deletion of the CCAAT-containing region caused a significant loss of promoter activity, an indication of cell-specific use of enhancer elements. Finally, a series of internal deletion and linker mutation studies of the -250 to -151 bp ovarian enhancer region revealed that the hMLH1 promoter contains multiple redundant enhancer elements capable of independent promoter activation and may explain the association of this region with methylation silencing of hMLH1.
Immunohistochemical analysis confirmed the presence of MLH1 protein in A2780 ovarian cancer cells and its absence in this
same cell line on acquired resistance to cisplatinum (A2780/CP). Transfection ...of a −1781-bp hMLH1 promoter construct into either A2780 or A2780/CP cells produced similar (30-fold) induction of luciferase, an indication
that the transcriptional machinery for hMLH1 expression remains intact. hMLH1-luciferase activity was also unaffected by re-expression of hMLH1 following treatment of A2780/CP cells with the methylase inhibitor 2′-deoxy-5-azacytidine. Serial 5′-deletion studies of
the hMLH1 promoter region in ovarian cancer cells localized transcriptional enhancers to a region (−250 to −151 bp) that excludes the
previously identified CCAAT element (−282) active in HeLa cells. When these same deletion constructs were transfected into
HeLa cells, deletion of the CCAAT-containing region caused a significant loss of promoter activity, an indication of cell-specific
use of enhancer elements. Finally, a series of internal deletion and linker mutation studies of the −250 to −151 bp ovarian
enhancer region revealed that the hMLH1 promoter contains multiple redundant enhancer elements capable of independent promoter activation and may explain the association
of this region with methylation silencing of hMLH1 .
Inhibitors of histone deacetylase activity are emerging as a potentially important new class of anticancer agents. In the
current studies, exposing A2780 ovarian cancer cells to the histone ...deacetylase inhibitor trichostatin A (TSA) produced a
marked change in cellular morphology, proliferation, and differentiation. Within 24 h of TSA treatment, there was a morphological
transformation of the cells, with increased cytoplasm, a more epithelial-like columnar appearance, and the emergence of distinct
cellular boundaries. Commensurate with the morphological transformation, TSA also inhibited cell proliferation; cells treated
with TSA for 72 h increased to 110% of the initial cell numbers versus control cell numbers of 622%, with a corresponding reduction in mitotic activity and a flow cytometry S-phase fraction of
3.9% in TSA-treated cells versus 28.8% for control. TSA also induced epithelial-like differentiation with increased cytokeratin expression from 2% of controls
to 22–25% of TSA-treated cells and the reappearance of intercellular plasma membrane junctions and primitive microvilli. Immunocytochemical
analyses indicate the molecular mechanism underlying the actions of TSA on A2780 cell cycle progression and differentiation
involves reexpression of the CDK inhibitor p21. Elevated levels of p21, in TSA-treated cells, were associated with a reduction
in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). TSA also caused a decrease in the helix-loop-helix
inhibitor of differentiation/DNA binding protein Id1, with no change in Id2 levels. In conclusion, the observed TSA-induced
changes in p21, Rb, and Id1 are consistent with cell cycle senescence and differentiation of A2780 ovarian cancer cells.
Biochemical methods that accurately detect ischemic damage in livers stored by flush cooling would be useful in the efficient development of new storage solutions. This study compares UW and Collins' ...solutions to evaluate those biochemical parameters which may be useful in assessing the reversibility of ischemic damage and the efficiency of organ storage solutions. Livers stored in UW solution showed higher levels of adenine nucleotides at all storage times studied. This increase in adenine nucleotides averaged 29% and was statistically significant. There was also a significant increase in the NAD levels in organs stored in UW solutions; however, the ATP levels were not significantly different after storage in either solution. Storage of livers in UW solution also decreased the amount of DNA damage which occurs with storage as compared to storage in Collins' solution, becoming statistically significant after 48 hr of storage. This suggests that these biochemical parameters may be useful in designing improved storage solutions since they reflect the extent of ischemic damage to the organ and can be determined on a small section of liver taken by biopsy.
The afterhyperpolarization (AHP) that follows repetitive stimulation was recorded intracellularly from CA1 pyramidal neurons in the guinea pig hippocampal slice preparation. Although the late AHP ...could be blocked by histamine (1-10 microM), forskolin (10 microM) and 8-bromo-cyclic AMP (100 and 500 microM), neither prostaglandins D2, E1 and F2 alpha (0.5 microM) nor vasoactive intestinal polypeptide (0.5 microM) had any effect on the AHP, membrane potential, membrane resistance or action potential properties.
The objective of this study was to characterize the previously demonstrated decrease in the molecular weight of DNA from kidneys submitted to storage injury. DNA from kidneys stored at either 0 ...degrees C for 24-96 hr or 37 degrees C for 1-3 hr underwent limited hydrolysis when incubated with S1 nuclease, an enzyme which specifically degrades single-stranded DNA. For warm-storage injury (37 degrees C), the susceptibility of the DNA toward S1 nuclease hydrolysis increased progressively with storage time. In the case of cold-storage injury (0 degrees C), a maximum degree of single strandedness was observed in the DNA representing 60 hr of storage. DNA from kidneys stored for 72 hr or longer was a poor substrate for S1 nuclease. Additionally, the nucleotide composition of the single-stranded regions was analyzed by high-performance liquid chromatography (HPLC). The results showed that single-stranded regions initially rich in dA and dT are formed during warm-storage injury. No such favoritism for a particular base was observed in single-stranded regions produced during cold-storage injury. The data suggest that both warm- and cold-storage injury promote DNA degradation. The storage temperature apparently dictates the mechanism(s) by which the degradative process proceeds.
The objective of this study was to measure the effect of diet on the distribution of RNA polymerase activity in mice kidney. Bound RNA polymerase activity was decreased in kidneys of fasted mice. ...During a short fast (16 hours), this decrease in bound RNA polymerase I activity was accompanied by an increase in free activity and suggested a possible interference in binding as the reason for decreased bound activity. Bound RNA polymerase II also decreased but with no increase in free activity. During longer fast times (40 hours), the bound RNA polymerase activity (RNA polymerase I and II) remain depressed and the free activity returns to control levels. Fasting decreased bound RNA polymerase I in liver by 41% after 40 hours but had little effect on the free activity. Liver of mice, fed a protein-free diet, were found to have an increased RNA polymerase activity due to a large increase in free RNA polymerase I activity. All other liver RNA polymerase activities were unchanged. In contrast, kidney's of mice fed a protein-free diet, have decreased RNA polymerase activity due to a decrease both in bound RNA polymerase I and II; free RNA polymerase is unchanged. These results demonstrate that diet has a very selective effect on RNA polymerase activity.
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