Damage and Alterations in Gene Expression Breast Tumor Cell Line: Evidence for Induction of Gene-specific Antiproliferative Activity of Teniposide (VM-26) in the MCF-7 Dissociation between Bulk Damage to DNA and the Updated Version

In the MCF-7 breast iunior cell line, induction of bulk damage to DNA (measured either as total strand breaks or as double-strand breaks I fails to correspond »¡Hi the antiprciliferative activity of the demethylepipodophyllotoxin derivative, VM-26. In contrast, VM-26 produces an early (within 2-3 h) concentration-dependent reduction in e-myc expression (and of UNA synthesis) which parallels inhibition of cell growth, suggest ing the possibility of effects of VM-26 at the level of genomic regions which regulate DNA replicative function. Although VM-26 also produces a re duction in c-myc expression in K562 human leukemic cells, these alter ations fail to correspond with the concentration-dependent effects on cell growth in this cell line. Utilizing the newly developed alkaline unwinding/ Southern blotting assay in the MCF-7 breast tumor cell line, it was de termined that VM-26 induces damage within regions surrounding the C-HIVCgene and the ß-globin gene which exceeds that induced in both «-satelliti1 DNA and in LI repeat sequences; damage within c-myc and ß-globinalso exceeds that observed throughout the genome as a whole. These findings indicate that certain genomic regions incur preferential damage in MCF-7 cells exposed to VM-26. It appears possible that damage within such genomic regions could lead to alterations in expression of select genes associated with regulation of cellular proliferation, resulting in reduced DNA synthesis, compromised cell growth, and, ultimately, cell death. The present studies demonstrate that in the MCF-7 human breast iunior cell line, the induction of bulk damage to DNA by the topoisomerase II inhibitor VM-26 fails to correspond with inhibition of cell growth. In contrast, there appears to be a close correlation between growth inhibition und the inhibition of DNA synthesis, suggesting that perturbation of DNA synthesis may be an early biochemical effect of this antineoplastic drug. Consistent with these observations. VM-26 also produces an early, concentration-dependent reduction in the ex pression of the c-mvr oncogene which corresponds with the ultimate inhibition of growth in MCF-7 cells (but not in human K562 leukemic cells). In order to address the possibility that damage within select ge nomic regions may contribute to the antiproliferative activity of topoisomerase II inhibitors (16-18), an assay was developed (alkaline unwinding/Southern blotting) with the capacity to assess damage within large scale regions surrounding genes of interest (19). Using this assay, it was determined that VM-26 produces differential damage in the regions surrounding select genes in the MCF-7 breast tumor cell line. Therefore, the induction of such damage by topoisomerase II inhibitors may act as a signal leading to alterations in the expression of genes, such as c-myc, which are involved in the regulation of DNA synthesis (20. 21) and cell proliferation (22). INTRODUCTION The demethylepipodophyllotoxin derivative, VM-26.' is one of a number of drugs which have been shown to interfere with the religation activity of DNA topoisomerase II via stabilizaiion of the DNAtopoisomerase II complex (1-3), resulting in the production of DNA strand breaks (4. 5). Stabilization of the "cleavable complex" and the concomitant induction of DNA strand breaks have been shown to correspond closely with drug cytotoxicity and/or antiproliferative ac tivity (6-8). suggesting that these lesions in bulk DNA mediate the antitumor effects of topoisomerase II inhibitors. In contrast, in some tumor cell lines, various topoisomerase II inhibitors have been shown to express cytotoxicity which fails to correspond with damage to bulk DNA (9-15), suggesting that bulk DNA damage is an incomplete explanation for drug action. Received 2/16/93; accepted 5/21/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by a grant from the Thomas F. and Kate M. Jeffress Trust, a Grant-In-Aid to faculty at the Medical College of Virginia, a grant from the AD Williams Foundation. NIH Grants CA-40615 and CA-51657. ACS Grant DHP-49. and a Pittsburgh Cancer Institute Postdoctoral Fellowship (M. K. R.). 2 To whom requests for reprints should be addressed. 'The abbreviations used are: VM-26 (teniposide). 4'-demethylepipodophyllotoxin-4(4.6-O-thenylidene-/3-n-glucopyranoside); AUSB; alkaline unwinding/Southern blotting assay: MTT. 3-(4.5-dimethylthiazol-2-yll-2.5-diphenyltetrazolium bromide: DMSO. di methyl sulfoxide: SDS. sodium dodecyl sulfate; TCA. trichloroacetic acid; dsDNA. dou ble-stranded DNA; crdsDNA, percentage of total DNA remaining double-stranded (acidprecipitable DNA/total DNA X 100); Fjs, fraction of hybridization sequence remaining double stranded, compared to control (density of hybridization signal of treated sample/ control); DMEM. Dulbecco's modified Eagle's medium: PBS. phosphate-buffered saline. MATERIALS AND METHODS