Jana L. Gump

Histone deacetylase interacts directly with DNA topoisomerase II

Histone deacetylases (HDACs) modify nucleosomal histones, have a key role in the regulation of gene transcription, and may be involved in cell-cycle regulation, differentiation and human cancer. Purified recombinant human HDAC1 protein was used to screen a cDNA expression library, and one of the clones identified encoded DNA topoisomerase II (Topo II), an enzyme known to have a role in transcriptional regulation and chromatin organization. Coimmunoprecipitation experiments indicate that HDAC1 and HDAC2 are associated with Topo II in vivo under normal physiological conditions. Complexes containing Topo II possess HDAC activities, and complexes containing HDAC1 or HDAC2 possess Topo II activities. HDAC and Topo II modify each others activity in vitro and in vivo. Our results indicate the existence of a functionally coupled complex between these two enzymes and offer insights into the potential mechanisms of action of both enzymes.

Effects of wild‐type p53 expression on the quantity and activity of topoisomerase IIα and β in various human cancer cell lines

The p53 null HL‐60 cell line was transfected with plasmids coding for either the wild‐type p53 or mutant p53 gene. The stable expression of wild‐type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR‐529. HL‐60 cells expressing wild‐type p53 demonstrated 8‐ to 10‐fold more VP‐16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild‐type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild‐type p53 in the K562 cell line resulted in a 3‐fold increase in sensitivity to VP‐16. The quantity of topoisomerase IIα was not altered by the transfection as determined by immunoblotting, while the amount of the β isoform was increased 2.5‐fold in HL‐60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIα was substantially diminished in both stable and inducible wild‐type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP‐16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIα. No changes in topoisomerase IIβ immunostaining were observed. These results suggest that an epitope for topoisomerase IIα is concealed in cells expressing wild‐type p53 and that a complex between topoisomerase IIα and p53 may be disrupted by the addition of antitumor drugs. J. Cell. Biochem. 75:245–257, 1999.

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase IIα associated with nuclear matrices from wild‐type and drug‐resistant Chinese hamster ovary cell lines

Topo IIα is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo IIα associated with the nuclear matrix in drug‐resistant SMR16 and parental cells in the presence and absence of VP‐16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole‐mount spreading of residual structures permits, by means of isoform‐specific antibody and colloidal‐gold secondary antibodies, an estimate of the amount of topo IIα in individual nuclear matrices. There are significant variations in topo IIα amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix–associated topo IIα than the resistant cell line matrices. Nuclear matrix–associated topo IIα from wild‐type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo IIα in the same matrix; in fact they were inversely related. In situ whole‐mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures. J. Cell. Biochem. 67:112–130, 1997.

Phase 2 study of gemcitabine and irinotecan in metastatic breast cancer with correlatives to determine topoisomerase I localization as a predictor of response

Gemcitabine incorporation into DNA enhances cleavage complexes in vitro when combined with topoisomerase I inhibitors and demonstrates synergy in cancer cells when given with irinotecan. Topoisomerase I inhibitors require that topoisomerase I interacts with DNA to exert activity.

Sequential oral 9-nitrocamptothecin and etoposide: A pharmacodynamic- and pharmacokinetic-based phase I trial

Purpose: Resistance to topoisomerase (topo) I inhibitors has been related to down-regulation of nuclear target enzyme, whereas sensitization to topo II inhibitors may result from induction of topo II by topo I inhibitors. Here, we evaluated a sequence-specific administration of a topo I inhibitor followed by a topo II inhibitor. Experimental Design: Twenty-five patients with advanced or metastatic malignancies were treated with increasing doses (0.75, 1.0, 1.25, 1.5, 1.75, or 2.0 mg/m2) of 9-nitrocamptothecin (9-NC) on days 1 to 3, followed by etoposide (100 or 150 mg/d) on days 4 and 5. At the maximally tolerated dose, 20 additional patients were enrolled. The median age was 60 years (range, 40–84 years). Endpoints included pharmacokinetic analyses of 9-NC and etoposide, and treatment-induced modulations of topo I and II expression in peripheral blood mononuclear cells. Results: Neutropenia, thrombocytopenia, nausea, vomiting, diarrhea, and fatigue were dose-limiting toxicities and occurred in six patients. Despite a median number of four prior regimens (range 1–12), 2 (4%) patients had an objective response and 13 (29%) patients had stable disease. In contrast to the expected modulation in topo I and IIα levels, we observed a decrease in topo IIα levels, whereas topo I levels were not significantly altered by 9-NC treatment. Conclusions: Sequence-specific administration of 9-NC and etoposide is tolerable and active. However, peripheral blood mononuclear cells may not be a predictive biological surrogate for drug-induced modulation of topo levels in tumor tissues and should be further explored in larger studies. [Mol Cancer Ther 2006;5(8):2130–7]

A phase i study of high-dose melphalan + topotecan + VP-16 phosphate (Mtv) Followed by autologous stem cell rescue in multiple myeloma

Abstract We conducted a dose-escalation trial of topotecan (TPT) given in combination with melphalan & VP-16. In this HDC protocol in multiple myeloma the drugs were given as melphalan (150 mg/m 2 , total dose)→TPT (0 to 27 mg/m 2 , total dose)→VP-16 phosphate (2400 mg/m 2 , etoposide equivalent total dose). Thirty-five patients have been enrolled in this study. Ten patients were enrolled in the first dose level (melphalan and VP-16 phosphate only), while TPT was dose-escalated from 10 mg/m 2 to 27 mg/m 2 (total dose over 3 days) in dose levels 2–5. The MTD of high-dose topotecan is 20 mg/m 2 , with a DLT or grade 3/4 mucositis and enteritis. The 100 day non-relapse mortality is 5.7%. There have been two regimen-related deaths; one patient died of cardiotoxicity and the other of sepsis/ARDS after being intubated for severe mucositis. The median day to an ANC >500/μl is day +10, while the median day to a platelet count >50,000/μl is day +17.5. The overall response in 31 evaluable patients is 52% (10 CR + 6 PR) while another 42% demonstrated stable disease. The event free survival and overall survival at 6 months are 77% ± 8% and 96% ± 3.5%, respectively. The PK of TPT in this study have been found to be similar to those found in a recent phase I study of ifosfamide→TPT→ etoposide, TIME regimen. The results of confocal microscopy analyses of malignant plasma cells obtained from MTV patients demonstrate that the subcellular distribution of topo I and IIα may be critical in determining the sensitivity of these cells to inhibitors of these enzymes.