Deletion at chromosome 3p21.3 is the earliest and the most frequently observed genetic alteration in lung cancer, suggesting that the region contains tumor suppressor gene(s) (TSG). Identification of those genes may lead to the development both of biomarkers to identify high-risk individuals and novel therapeutics. Previously, we cloned the H37/Luca15/RBM5 gene from 3p21.3 and showed its TSG characteristics. To investigate the physiologic function of H37 in the lung and its mechanism of tumor suppression, we have stably transfected H37 into A549 non-small cell lung cancer cells. A549/H37 cells show significant growth inhibition compared with the vector controls by in vitro and in vivo cell proliferation assays. Using this lung cancer cell model, we have found that the molecular mechanism of H37 tumor suppression involves both cell cycle (G 1 ) arrest and apoptosis. To further define H37's function in cell cycle/apoptotic pathways, we investigated differential expression profiles of various cell cycle and apoptosis regulatory proteins using Western blot analysis. Both cyclin A and phophorylated RB levels were decreased in H37-transfected cells, whereas expression of Bax protein was increased. Mitochondrial regulation of apoptosis further downstream of Bax was investigated, showing change in the mitochondrial membrane potential, cytochrome c release into the cytosol, and enhanced caspase-9 and caspase-3 activities. We also report that H37 may mediate apoptosis in a p53-independent manner, and Bax knockdown by small interfering RNA suggests Bax plays a functional role downstream of H37. Lastly, we proposed a tumor suppression model of H37 as a post-transcriptional regulator for cell cycle/apoptotic-related proteins.

Loss of heterozygosity (LOH) at chromosome 3p21.3 is one of the most prevalent genetic disturbances occurring at the earliest stage of tumor development for a wide variety of human cancers, culminated in lung cancer. The 19 genes residing at 3p21.3 have been vigorously characterized for tumor suppressor activity and gene inactivation mechanism because of their potentially significant merits of clinical applications. Many of these 19 genes have been shown to manifest various growth inhibitory properties, however none of them are inactivated by coding mutations in their remaining allele as in the Knudson's two- hits hypothesis. Thus far the most prevailing, alternative gene inactivation mechanism known for the 3p21.3 TSGs is epigenetic silencing by promoter hypermethylation. Previously, we have focused our investigation on one of the 19 genes at 3p21.3, H37/RBM5, and demonstrated its tumor suppressor activity both in vitro and in vivo as well as its mRNA/protein expression loss from the remaining allele in a majority of the primary lung tumors examined. The current study tested our hypothesis that the H37 inactivation in primary lung tumors may, as seen in most of the other 3p21.3 TSGs, be due to hypermethylation in its promoter CpG islands. Contrary to this most plausible postulation, however, we found no evidence of epigenetic gene silencing for the H37 TSG. Here we suggest some of the possible, further- alternative means of the H37 gene expression loss in tumor, including defects in transcription and post-transcriptional/translational modifications as well as mechanisms related to haploinsufficiency.

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