Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation.

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

Naturally occurring resistance of bone marrow mononuclear and metastatic cancer cells to anticancer agents.

Numerous cancer patients fail standard chemotherapy or develop resistance to chemotherapy during the course of treatment. The purpose of this study is to elucidate the overall response of cells obtained from cancer patients and from normal individuals to chemotherapeutic agents. We analysed the chemosensitivity of cancer cells derived from bone marrow and from pleural effusions or ascites fluids from patients with different cancers. Chemosensitivity to doxorubicin, cisplatin and paclitaxel was determined using the MTT assay. We also determined the response of bone marrow mononuclear (BMMN) cells. There was a wide range of responses to chemotherapy drugs in samples from different individuals. This was observed in cells derived from bone marrow and from ascites or pleural fluids. Large variations were also observed among morphologically normal BMMN cells and metastatic cancer cells from chemo-naïve patients. Cancer cells can easily be collected from ascites or pleural fluids and reliably assayed for chemosensitivity. We describe here that inherent chemoresistance may be a reason for the lack of response to chemotherapy in some patients. We discuss the potential of using the determination of natural resistance to dictate the drugs to be employed for treatment.

Flavopiridol sensitivity of cancer cells isolated from ascites and pleural fluids.

PURPOSE: We examined the efficacy of flavopiridol, a cyclin-dependent kinase inhibitor that is undergoing clinical trials, on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers. EXPERIMENTAL DESIGN: Metastasized cancer cells were isolated from the pleural fluids (n = 20) or ascites (n = 15) of patients, most of whom were refractory to chemotherapy. These primary cancer cells were used within 2 weeks of isolation without selecting for proliferative capacities. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay was used to characterize the response of these cancer cells to commonly used chemotherapeutic agents, and their response to flavopiridol was compared with rapidly dividing cultured cell lines. RESULTS: The primary cancer cells displayed phenotypes that were different from established cell lines; they had very low replication rates, dividing every 1 to 2 weeks, and underwent replicative senescence within five passages. These primary tumor cells retained their resistance to chemotherapeutic drugs exhibited by the respective patients but did not show cross-resistance to other agents. However, these cancer cells showed sensitivity to flavopiridol with an average LD50 of 50 nmol/L (range, 21.5-69 nmol/L), similar to the LD50 in established cell lines. Because senescent cells also showed similar sensitivity to flavopiridol, it suggests that the mechanism of action is not dependent on the activity of cyclin-dependent kinases that regulate the progression of the cell cycle. CONCLUSION: Using cancer cells isolated from the ascites or pleural fluids, this study shows the potential of flavopiridol against cancer cells that have developed resistance to conventional chemotherapeutic agents.

H1 family histones in the nucleus. Control of binding and localization by the C-terminal domain.

H1 histones bind to DNA as they enter and exit the nucleosome. H1 histones have a tripartite structure consisting of a short N-terminal domain, a highly conserved central globular domain, and a lysine-and arginine-rich C-terminal domain. The C-terminal domain comprises approximately half of the total amino acid content of the protein, is essential for the formation of compact chromatin structures, and contains the majority of the amino acid variations that define the individual histone H1 family members. This region contains several cell cycle-regulated phosphorylation sites and is thought to function through a charge-neutralization process, neutralizing the DNA phosphate backbone to allow chromatin compaction. In this study, we use fluorescence microscopy and fluorescence recovery after photobleaching to define the behavior of the individual histone H1 subtypes in vivo. We find that there are dramatic differences in the binding affinity of the individual histone H1 subtypes in vivo and differences in their preference for euchromatin and heterochromatin. Further, we show that subtype-specific properties originate with the C terminus and that the differences in histone H1 binding are not consistent with the relatively small changes in the net charge of the C-terminal domains.

TBP-associated factor 1 overexpression induces tolerance to Doxorubicin in confluent H9c2 cells by an increase in cdk2 activity and cyclin E expression.

Doxorubicin (DOX) is a DNA topoisomerase II inhibitor widely used in anticancer treatment, however, it can lead to irreversible cardiac damage with severe debilitation. TBP-binding associated factor 1 (TAF1) is increased in DOX damaged hearts in vivo and in cardiomyocytes in vitro. To identify the functional role for TAF1 in DOX-treated heart we overexpressed wild type and mutant TAF1 in H9c2 cells. Overexpression of wild-type TAF1, but not N-terminal kinase domain mutants, increased tolerance to DOX in confluent cells. DOX treatment can cause prolonged G1 arrest. We found increased cdk2 activity coupled to increased cyclin E protein and decreased p21(waf1Cip1) and p27(Kip1) protein to correlate only with increased DOX tolerance and wild-type TAF1. DOX sensitivity was restored when the cdk2-inhibitor Roscovitine was co-administered with DOX. Overexpression of cdk2-alone increased resistance to DOX. Thus, TAF1 induced DOX tolerance in confluent cells through an increase in cdk2 activity is directed by the TAF1 N-terminal domain. These studies suggest new avenues for myocardial protection against DOX toxicity and suggest a role for cdk2 in chemorefractory cells.

The C-terminal domain is the primary determinant of histone H1 binding to chromatin in vivo.

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1 in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 binding in vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.