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Loss of amplified genes by poly(ADP-ribose) polymerase inhibitors
M Nagao1, M Nakayasu, S Aonuma
1Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.
Abstract:
A poly(ADP-ribose) polymerase inhibitor, benzamide (BA), was found to induce flat revertants of NIH 3T3 cells that had been transformed by human Ha-ras, rat Ki-ras, rat c-raf, and human ret-II. These genes had been amplified in original transformants, but they were completely eliminated by BA. Contrary to this, endogenous activated Ha-ras in a human bladder carcinoma cell line, T24, was not eliminated by BA. The gene loss seemed to be restricted to exogenous and/or amplified sequences. BA also eliminated the amplified c-myc gene in HL-60 cells, concomitant with differentiation into granulocytes. We demonstrated that the amplified c-myc gene was not present as episomes. It is probably present as double minutes or a homogeneously staining region. Dimethylsulfoxide also induced differentiation at a concentration that did not inhibit poly(ADP-ribose) polymerase. The cell lost the c-myc gene in association with this differentiation. The amplified c-myc gene in a colon adenocarcinoma cell line, COLO 320HSR, and the amplified mdr-1 gene in an adriamycin-resistant myelogenous leukemia cell line, K562/ADM, were not eliminated by BA. Various poly(ADP-ribose) polymerase inhibitors also eliminated human Ha-ras in the NIH 3T3 transformant and the c-myc gene in HL-60 cells.
Insights
Benzamide, a poly(ADP-ribose) polymerase inhibitor, eliminates amplified oncogenes like Ha-ras and c-myc in specific cell lines, potentially reversing transformation and inducing differentiation.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Certain oncogenes, when amplified, can drive cellular transformation and cancer progression.
- Poly(ADP-ribose) polymerase (PARP) inhibitors are explored for their therapeutic potential in cancer.
- The mechanisms by which gene amplification contributes to cancer and how it can be reversed are areas of active investigation.
Purpose of the Study:
- To investigate the effect of benzamide (BA), a poly(ADP-ribose) polymerase inhibitor, on amplified oncogenes in transformed cells.
- To determine if BA can induce phenotypic reversion and/or gene loss in cancer cell lines with amplified genes.
- To explore the relationship between PARP inhibition, gene elimination, and cellular differentiation.
Main Methods:
- Treatment of NIH 3T3 cells transformed by various oncogenes (Ha-ras, Ki-ras, c-raf, ret-II) with benzamide.
- Analysis of gene copy number and expression in treated cells.
- Investigating the effect of benzamide on endogenous activated Ha-ras in T24 cells.
- Examining benzamide's impact on amplified c-myc in HL-60 cells and amplified c-myc/mdr-1 in COLO 320HSR and K562/ADM cells, respectively.
- Utilizing dimethyl sulfoxide as a comparative inducer of differentiation.
Main Results:
- Benzamide induced flat revertants in NIH 3T3 cells transformed by exogenous oncogenes, with complete elimination of amplified Ha-ras, Ki-ras, c-raf, and ret-II.
- Endogenous activated Ha-ras in T24 cells was not eliminated by benzamide, suggesting gene loss is restricted to exogenous/amplified sequences.
- Benzamide eliminated the amplified c-myc gene in HL-60 cells, coinciding with differentiation into granulocytes.
- The amplified c-myc gene in HL-60 cells was not present as episomes but likely as double minutes or homogeneously staining regions.
- Dimethyl sulfoxide also induced c-myc gene loss and differentiation in HL-60 cells.
- Amplified c-myc in COLO 320HSR and amplified mdr-1 in K562/ADM cells were not eliminated by benzamide.
- Various PARP inhibitors demonstrated similar effects on Ha-ras and c-myc gene elimination.
Conclusions:
- Benzamide and other PARP inhibitors can selectively eliminate amplified exogenous oncogenes, leading to phenotypic reversion in transformed cells.
- The gene-eliminating effect of PARP inhibitors appears specific to amplified sequences, not endogenous activated oncogenes.
- PARP inhibition can be linked to cellular differentiation, as observed with c-myc elimination in HL-60 cells, suggesting a role in modulating gene expression and cell fate.