Studies Of Tumor Suppressor Gene ING1 In Lung Carcinoma

Inhibitor of growth 1, ING1, the member of the ING tumor suppressor family, was discovered in 1996. It was isolated using a cloning strategy based on suppressive subtractive hybridization and the theory that the activities expression of tumor suppressor genes are selectively suppressed or reduced in tumor cells but maintained or elevated in normal senescent cells. ING1 plays a significant role in multiple cellular activities such as growth regulation, apoptosis, senescence and DNA repair upon UV-induced DNA damage which are characteristics typical of tumor suppressors. ING1 is down-regulated in several tumor types, including breast, gastric, esophageal, blood, lung and brain. ING1 gene mutation is rare or nonsense in cancer, while loss of heterozygosity (LOH) is common in cancer. ING1 shares many of the biological effects seen in response to p53, raising the possibility that the biochemical functions of p33^ING1b and p53 may be interrelated which is considered the mediation by the structures of plant homeodomain (PHD), nuclear localization sequences (NLS ) and nucleolar targeting sequences(NTS); the structures also mediate the combination with MDM2, PCNA, C-myc, Bax etc.Lung cancer is the commonest cause of cancer death among men and women in the world especially in the developed countries and the developing country will reach that level before long. The most important and cost-effective management for lung cancer is smoking cessation, but for those with the disease, novel agents and treatment approaches are urgently needed.Gene therapy is an exciting prospect for treatment and potential cure of lung cancer because genes can be transduced into cancer cells to correct genetic lesions such as replacing defective tumor suppressor genes or inactivating oncogenes. p53 is noticed and studied extremely, but it is not acting independently, p33^ING1b protein is newly founded molecular companion in inducing cell cycle arrest and apoptosis.To detect the abnormal expression of p33^ING1b, the main product of ING1 gene, two hundreds and seventeen cases of formalin-fixed, paraffin-embedded specimens were examined for p33^ING1b protein expression by immunohistochemistry using tissue microarray. To investigate the relationship between p33^ING1b and p53, PCNA, the expression of p53 and PCNA were detected by immunohistochemistry too. p33^ING1b expression was found in 102 of 217 carcinomas(47.0%) totally, and was found in 66.7%(62/93), 33.3%(23/69), 37.9% (11/29), 15.4%(2/13), 44.4%(4/9), 0%(0/3) and 0%(0/1) of squamous carcinoma(SCC), adenocarcinoma (AC), bronchioloalveolar carcinoma (BAC), adenosquamous carcinoma (AdCa), small cell lung carcinoma(SCLC), carcinoid and mucoepidermoid carcinoma respectively. The p33^ING1b positivity in SCC was significantly higher (P<0.05) than AC, BAC, and AdCa, while there was no significantly difference between other types.Secondly, to detect the alteration of ING1 gene in lung caner, we analysis the loss of heterozygosity of four microsatellites of ING1 gene, Seventy-six cases of fresh-frozen lung cancers were analysis with polymerase chain reaction-single trand conformation polymorphism(PCR-SSCP). LOH was found in 39 of 70 lung carcinomas (55.7%) on four ING1 loci and found in 14, 17, 15, 28 carcinomas on D13S261, D13S1047, D13S1315 and DS42490 respectively. LOH on DS42490 located in the intron of ING1 gene was significantly higher than the other loci (P<0.05), but the LOH frequency was not correlation with clinicopathological characteristics (P>0.05). The p33^ING1b expression was not correlated with the clinicopathological characteristics but there was a highly significant inverse correlation with LOH frequency (P<0.05).Thirdly, to investigate the inhibitory effect of tumor ING1, recombinant ING1b plasmids were transfected into lung cancer cell lines A549 and SK-MES-1, then cell cycle, apoptosis, cell growth rate and the protein expression were analyzed after transfected. The eukaryotic expression plasmids led the overexpression of protein, and inhibited the growth of the A549 and SK-MES-1 cell lines both, induced cell cycle arrest and more apoptosis. Combined transfection of ING1b and wtp53 promoted the cell cycle arrest, higher apoptosis rate and elevated expression of p21WAF1. Co-immunoprecipitation detected the combination of p33^ING1b and wtp53 in the combined transfected cell of ING1b and wtp53, while the combination was not exised in the lung carcinoma tissues.Conclusion: The loss of heterozygosity of four microsatellites of ING1 gene and loss or reduced expression of it's mainly product may be an important cause of carcinogenesis. The ING1b gene can inhibit the growth of the lung cancer cell lines and induced cell cycle arrest and apoptosis, and p53 can promote this function through the combination with p33^ING1b and upregulation the p21WAF1.