Loss of the MYC gene amplified in human HL-60 cells after treatment with inhibitors of poly(ADP-ribose) polymerase or with dimethyl sulfoxide.

Abstract

In HL-60 cells, a human promyelocytic leukemia cell line, the human c-myc gene, designated MYC, is amplified about 16-fold. On differentiation of the HL-60 cells into granulocytes induced by several inhibitors of poly(ADPribose) polymerase [NAD' poly(adenosine diphosphate Dribose)ADP-D-ribosyltransferase, EC 2.4.2.30] including benzamide, nicotinamide, coumarin, and 4-hydroxyquinazoline or dimethyl sulfoxide, some MYC loss was observed. In contrast, benzoic acid, a noninhibitory analogue of benzamide, did not induce either granulocytic differentiation or loss of MYC. Loss ofMYC seems to be associated with granulocytic differentiation because the time course of its loss was similar to that of appearance of nitroblue tetrazolium-positive cells, mature granulocytes, and its loss was not observed on differentiation of HL-60 cells into macrophages induced by phorbol 12-myristate 13-acetate or teleocidin. The loss of MYC is not the reason for the down regulation of MYC expression observed within 1 hr after addition of inducers, since the loss of MYC was not detected by 1-day treatment with inducers.Poly(ADP-ribose) polymerase [NAD+ poly(adenosine diphosphate D-ribose)ADP-D-ribosyltransferase, EC 2.4.2.30] synthesizes protein-bound polymers of ADP-ribose using NAD as a substrate (1). Nuclear proteins, such as histone H1 and several nonhistone chromatin proteins are poly(ADPribosyl)ated (2). Poly(ADP-ribosyl)ation has been suggested to be involved in DNA repair, DNA replication, sister chromatid exchanges, and cell differentiation (2). Recently we have reported that benzamide and other inhibitors of poly(ADP-ribose) polymerase induce the efficient loss of transfected amplified oncogenes from NIH 3T3 transformants (3). This loss of transfected amplified genes was observed with several oncogenes, including c-ras family genes, ret, a tyrosine kinase, and c-raf, a serine/threonine kinase. It is not known yet whether the deletion occurred because those genes are exogenous or amplified.In the present study, we examined the effect of poly(ADPribose) polymerase inhibitors on HL-60 cells focusing on the endogenous amplified human c-myc gene, MYC. HL-60 cells were isolated from a promyelocytic leukemia patient (4). Fresh, uncultured leukemia cells from which HL-60 cells were isolated were reported to show 16-to 32-fold amplification of MYC (4). HL-60 cells are known to differentiate into granulocytes on treatment with poly(ADP-ribose) polymerase inhibitors (5). We found that inhibitors of poly(ADPribose) polymerase caused efficient loss of the MYC gene in HL-60 cells and concomitantly induced granulocytic differentiation. Dimethyl sulfoxide, which is well known to induce granulocytic differentiation of HL-60 cells, also induced loss of MYC.Amplified MYC genes in HL-60 cells are reported to be located in homogeneously staining regions (6), double minute chromosomes (7), or episomes (8). We analyzed the structure of amplified MYC in HL-60 cells of our stock by pulsed-field gradient gel electrophoresis (PFGE) to understand t...

Granulocytic Differentiation of HL-60 Cells, both Spontaneous and Drug-Induced, Might Require Loss of Extrachromosomal DNA Encoding a Gene(s) Not c-MYC

“…Previously, Shima et al observed a loss of c-MYC copies during treatment of HL-60 cells with DMSO and suggested this as a cause of differentiation (18). We reported that the spontaneous granulocytic differentiation of HL-60 cells that occurs without treatment with drugs was also associated with loss of c-MYC copies and it progressed at a similar slow rate as the DMSOinduced differentiation (19).…”

Poly(ADP‐ribose) and carcinogenesis

“…Exogenously introduced oncogenes, H-ras T24 , Ki-ras, N-ras, c-raf, and c-ret-II, in NIH 3T3 transformants were lost by treatment with PARP inhibitors (Nakayasu et al, 1988), and the endogenously amplified c-myc gene in HL-60 cells was also lost by a similar treatment (Shima et al, 1989). It is not clear yet whether dysfunction of PARP-1 is the sole cause, or whether other members of the PARP family, mentioned below, may also contribute to this phenomenon.…”

Extrachromosomal Double Minutes and Chromosomal Homogeneously Staining Regions as Probes for Chromosome Research

“…The HU treatment reduced the number of DMs in advanced ovarian carcinoma in vivo [Raymond et al, 2001]. The inhibitor of poly(ADP-ribose) polymerase or dimethyl sulfoxide also reduced the copy number of amplified c-myc in HL-60 cells [Shima et al, 1989]. Furthermore, ionizing radiation accelerated the loss of amplified MDR1 on DMs in multi-drug resistant KB cells [Sanchez et al, 1998;Schoenlein et al, 2003] or amplified c-myc in COLO 320DM cells [Schoenlein et al, 2003].…”