Molecular cloning, characterization and isolation of novel spliced variants of the human ortholog of a rat estrogen-regulated membrane-associated protein, UO-44.

We have previously reported the characterization of an estrogen-regulated rat uterine-ovarian-specific complementary DNA (UO-44). To understand the involvement of this protein in the initiation and progression of human ovarian and uterine cancers, we now report the cloning and characterization of the human ortholog (HuUO-44). HuUO-44 is mapped to chromosome 10q26.13 and contains nine exons. Multiple tissue Northern blot detected two HuUO-44 transcripts of approximately 2 and 3 kb in the pancreas. RT-PCR demonstrated that HuUO-44 undergoes a complex series of alternative splicing events between exons 2 and 6 that yielded four novel splice variants, HuUO-44A, HuUO-44B, HuUO-44C and HuUO-44D. Putative functional motifs identified in HuUO-44 are two CUB domains and a zona-pellucida domain. Transfection studies demonstrated the membrane-associated nature of HuUO-44. By immunohistochemistry, HuUO-44 was located to the normal ovarian and ovarian tumor epithelial cells; in NIH-OVCAR3 ovarian cancer cells, HuUO-44 was detected only at the leading edge of the dividing cells. Most importantly, a marked loss in cell attachment and proliferation was observed in NIH-OVCAR3 cells cultured in the presence of a polyclonal HuUO-44 antiserum. These findings suggest the potential role of HuUO-44 in cell motility, cell-cell interactions and/or interactions with the extracellular matrices.

Structural characterization of three novel rat OKL38 transcripts, their tissue distributions, and their regulation by human chorionic gonadotropin.

We previously identified a novel pregnancy-induced growth inhibitory gene, OKL38. To develop a rat model for further characterization of OKL38's role in the initiation and progression of breast and ovarian cancer, we now report the cloning and characterization of three novel rat OKL38 cDNAs that are derived through alternative splicing and differential promoter usage. These three transcripts differ in their 5' untranslated regions but share a common open reading frame that encoded for a 52-kDa protein. OKL38 is mapped to chromosome 19, spanning a region of approximately 15 kb, and contains eight exons. Differential expression of these three rat OKL38 transcripts was observed in liver, kidney, ovary, mammary gland, and uterus. In situ hybridization localized the rat OKL38 transcripts to the luminal epithelial cells of the rat mammary gland and to the granulosa cells in the rat ovary. In vivo studies showed that the RtOKL38-2.0 transcript and protein were regulated by human chorionic gonadotropin in the rat mammary gland and ovary. Importantly, overexpression of RtOKL38-enhanced green fluorescence protein fusion protein in Buffalo rat liver cells resulted in growth inhibition and cell death. Our present findings suggest that OKL38 may function as an effector for human chorionic gonadotropin protection against mammary carcinogenesis, and the availability of the three rat OKL38 cDNAs may help to elucidate the possible role of OKL38 in cellular growth, differentiation, and carcinogenesis.

Quercetin-induced growth inhibition and cell death in nasopharyngeal carcinoma cells are associated with increase in Bad and hypophosphorylated retinoblastoma expressions.

Nasopharyngeal carcinoma is a common cancer in South-East Asia, especially among people of Chinese origin. In this report, we investigate the effects of quercetin on the growth of wild-type and mutant p53 nasopharyngeal carcinoma cell lines, HK1 and CNE2 respectively. The wild-type p53 HK1 was more susceptible to growth inhibition by quercetin than the mutant p53 CNE2. The ID50 values for HK1 and CNE2 were 35.0 and 54.5 microM respectively. Cell growth arrest was initiated by the up-regulation of retinoblastoma gene expression, resulting in cell cycle arrest in either the G2/M or G0/G1 phase at 14.8 and 52.1 microM quercetin respectively regardless of the p53 status. Flow cytometry experiments revealed that quercetin-induced apoptosis during the first 24 h followed by necrosis in both HK1 and CNE2. Western blot experiments confirmed that cytotoxic killing of HK1 and CNE2 by quercetin was mediated by the up-regulation of pro-apoptotic protein Bad, caspase-3 and -7, resulting in cell death by apoptosis. Our study demonstrates that quercetin inhibits cell growth of nasopharyngeal carcinoma cell lines HK1 and CNE2 by inhibiting cell cycle progression to S phase. Quercetin is also able to induce apoptosis and necrosis in these cells regardless of the p53 status.

Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost.

In a search for potential ethanologens, waste compost was screened for ethanol-tolerant thermophilic microorganisms. Two thermophilic bacterial strains, M5EXG and M10EXG, with tolerance of 5 and 10% (v/v) ethanol, respectively, were isolated. Both isolates are facultative anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative, Gram-negative rods that are capable of utilizing a range of carbon sources including arabinose, galactose, mannose, glucose and xylose and produce low amounts of ethanol, acetate and lactate. Growth of both isolates was observed in fully defined minimal media within the temperature range 50-80 degrees C and pH 6.0-8.0. Phylogenetic analysis of the 16S rDNA sequences revealed that both isolates clustered with members of subgroup 5 of the genus Bacillus. G+C contents and DNA-DNA relatedness of M5EXG and M10EXG revealed that they are strains belonging to Geobacillus thermoglucosidasius. However, physiological and biochemical differences were evident when isolates M5EXG and M10EXG were compared with G. thermoglucosidasius type strain (DSM 2542(T)). The new thermophilic, ethanol-tolerant strains of G. thermoglucosidasius may be candidates for ethanol production at elevated temperatures.

Genomic structure of human OKL38 gene and its differential expression in kidney carcinogenesis.

We previously demonstrated the growth inhibitory property of OKL38 and its possible roles in mammary carcinogenesis. To further understand the regulation and roles of OKL38 in tumorigenesis we proceeded to clone and characterize the human OKL38 gene and three of its variants with transcripts of 1.9, 2.2, and 2.4 kb. The human OKL38 gene spans approximately 18 kb and contains 8 exons and 7 introns with exon size ranging from 92 to 1270 bp. RT-PCR and sequence analysis suggest that different transcripts were arrived through differential promoter usage and alternate splicing. Multiple Tissue Expression array (MTE) and Multiple Tissue Northern blot (MTN) indicated that OKL38 was ubiquitously expressed in all tissues with high expression in liver, kidney, and testis. The cancer profiling array (CPA) of paired normal/tumor cDNA showed that OKL38 mRNA was down-regulated in 70% (14 of 20) of kidney tumors. Western analysis revealed that the OKL38 protein was undetectable in 78% (7 of 9 pairs) of kidney tumor tissues. Immunohistological analysis showed that 64% (14 of 22) of kidney tumors were either lost or underexpressed OKL38 protein compared with the adjacent normal tissue. A transfection study using OKL38-eGFP recombinant construct showed that overexpression of the 52 kDa OKL38 protein in A498 cells resulted in growth inhibition and cell death. This study demonstrates the complex genomic structure of the OKL38 gene and its growth inhibitory and cytotoxic properties. Our data suggest the potential use of OKL38 in diagnosis, prognosis, and/or treatment of kidney cancer.