INCREASED GENETIC STABILITY OF HELA-CELLS AFTER CONNEXIN-43 GENE TRANSFECTION

(1)To test the hypothesis that intact gap-junctional intercellular com munication (GJIC) is necessary for genomic stability, we compared the spontaneous and chemically induced mutation frequencies in GJIC-profic ient and -deficient HeLa cells. Thus. we determined microsatellite ins tability and mutation frequency in the HPRT gene in parental HeLa cell s, which have no GJIC ability, and in HeLa cells in which GJIC was res tored by transfection with the connexin 43 (Cx43) gene, When HeLa cell s with (Cx43(+)) or without Cx43 gene (Cs43(-)) were treated with N-me thyl-N'-nitro-N-nitrosoguanidine (MNNG) or methylnitrosourea, the Cx43 (+) cells survived better than Cx43(-) cells, The mutation frequency a l CA repeats was measured with a shuttle vector; in the vector, the co ding region of the beta-galactosidase gene was rendered out of frame b y insertion of CA repeats, and the frame could be restored by insertio n or deletion mutations of the Cri repeats. The mutation frequency at CA repeats was 2-fold lower in Cx43(+) cells than in Cx43(-), both bef ore and after exposure to MNNG of methylnitrosourea (P < 0.05), The fr equency of spontaneous HPRT gene mutations, selected by their resistan ce to 6-thioguanine, was 3-fold lower in Cx43(+) cells than Cx43(-) ce lls, Similarly, the frequency of MNNG-induced HPRT mutations was signi ficantly higher in Cx43(-) cells (P < 0.001), Similar results were obt ained even when the mutant selection process was carried out in the pr esence of alpha-glycyrrhetinic acid, a long-term inhibitor of GJIC, su ggesting that the observed effect is not due to unwanted killing of ce lls by GJIC-mediated metabolic cooperation, Thus, our data demonstrate that HeLa cells transfected with the Cx43 gene become more resistant to spontaneous as well as chemically induced genetic changes.