Expression of hTERT mRNA in a mortal liver cell line during S phase without detectable telomerase activity.

Normal human liver cells have a limited capacity for proliferation due to telomere shortening, whereas immortalized cells prevent shortening of the 3' single strand telomeric repeat by expressing telomerases, including human telomerase reverse transcriptase (hTERT). The hTERT transcript contains three deletion sites that give rise to alternatively spliced variants (ASVs). Recently, hTERT expression was observed in cycling primary presenescent human fibroblasts, which were believed to lack hTERT expression and telomerase activity. hTERT mRNA was expressed in the synthesis (S) phase of the cell cycle. Although hTERT mRNA has eight isoforms, it is not known which of the hTERT ASVs are expressed in S phase. In order to determine the possible relationships between the cell cycle and ASV expressions, we measured the full-length isoform and ASVs of hTERT mRNA in a mortal liver cell line and immortal cell lines that were synchronized in S phase of the cell cycle. Using RT nested-PCR analysis, the full-length isoform and alpha-deletion ASV of hTERT were detected in the LI90 mortal liver cell line at points when cells in S phase represented >48% of the cell population without detectable telomerase activity. hTERT was always expressed in the HLE and Huh-7 hepatocellular carcinoma cell lines, regardless of the cell cycle. Our results suggest the possibility that telomerase is regulated in a cell cycle-dependent manner in normal liver cells.

Quantitation of O6-methylguanine-DNA methyltransferase gene messenger RNA in gliomas by means of real-time RT-PCR and clinical response to nitrosoureas.

1. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was measured in 50 malignant gliomas that had received 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after the resection of the tumor by real-time reverse transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. 2. The mean absolute value of MGMTmRNA normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 50 tumors was 1.29 x 10(4)+/- 1.28 x 10(4) copy/microg RNA (mean +/- SD). The amount of MGMTmRNA less than 6 x 10(3) copy/microg RNA was the most significant factor in predicting the initial effect of treatment with ACNU by multi-variant regression analysis (p = 0.0157). 3. These results suggest that quantitation of MGMTmRNA is the excellent method for predicting for the effect of ACNU in glioma therapy.

Identification of an alternative splicing transcript for the resistin gene and distribution of its mRNA in human tissue.

OBJECTIVE: Adipocytes secrete a number of molecules such as tumor necrosis factor-alpha, leptin and free fatty acids that can influence the ability of the body to metabolize glucose. Recently, a novel 12.5 kDa cysteine-rich protein, termed resistin, was shown to be secreted by adipocytes. Resistin expression was markedly induced during the conversion of 3T3-L1 cells to mature adipocytes. Expression of resistin has been studied in human, mouse and rat; however, sequence information about an alternative splicing variant (ASV) of resistin mRNA has not been reported. In the present study, we investigated the occurrence of a novel ASV of the resistin gene in human normal tissues. DESIGN AND METHODS: We identified a novel ASV of resistin mRNA in human lung tissue by RT-PCR analysis in human lung tissue. We then investigated a novel ASV of resistin mRNA by real-time PCR analysis in 26 different types of normal human tissues. RESULTS AND CONCLUSIONS: We identified a novel deletion variant of the resistin transcript in the normal human tissues. The deleted transcript of resistin was characterized by an in-frame deletion of 78 bp, corresponding to the complete loss of exon 2 (resistin delta2 ASV). Thus, resistin delta2 ASV causes protein truncation. Our results provide the basis for more detailed studies on the regulation of resistin activity, and should assist in the development of clinical trials with resistin for the central regulation of adipogenesis and adipocyte metabolism.

Differential alternative splicing expressions of telomerase reverse transcriptase in gastrointestinal cell lines.

Telomerase is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric TTAGGG repeats at eukaryotic chromosome ends. One of the human telomerase components is hTERT, which has three alternative spliced sites that introduce eight isoforms of hTERT mRNA. The expression of these isoforms in gastrointestinal cell lines is unknown. We developed a PCR-based assay for detecting these splicing variants. In gastric and hepatocellular carcinoma cell lines, the gamma deletion variant and its combination variants, alpha- and gamma-, beta- and gamma-, and alpha-, beta- and gamma-deletion variants were frequently detected, while they were not detected in colorectal carcinoma cell lines. Our results provide important information of use for more detailed studies on the regulation of telomerase activity.

Expression profile of a gamma-deletion variant of the human telomerase reverse transcriptase gene.

The human telomerase reverse transcriptase (hTERT) is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of chromosomes. The hTERT transcript has been shown to have two deletion type alternative splicing sites. One deletion site induces the alpha-deletion variant, lacking 36 bp from exon 6, and the other induces the beta-deletion variant, lacking 182 bp from exons 7 and 8. Here, we identified a novel deletion variant of the hTERT transcript in hepatocellular carcinoma cell lines. The deleted transcript was characterized by an in-frame deletion of 189 bp, spanning nucleotides 2710 to 2898, corresponding to the complete loss of exon 11 (gamma-deletion). The region lacking in the gamma-deletion lies within RT motifs D and E, suggesting that it is missing conserved residues from the catalytic core of the protein. Both gamma- and alpha-deletion variants were occasionally detected, but the beta-deletion variant was frequently observed. Our results may provide important information for more detailed studies on the regulation of telomerase activity.

Genetic detection for hematogenous micrometastasis in patients with various types of malignant tumors using Uroplakin II derived primers in polymerase chain reaction.

The presence of circulatory metastasis is one of the most significant factors for poor-prognosis in patients with several types of cancer. To establish a sensitive reverse transcription PCR assay to detect micrometastasis in blood containing several cancer types, we first investigated Uroplakin II (UP II), a novel molecular marker for human transitional cell carcinoma of the bladder, in 25 types of normal organs. In our study, UP II mRNA was detected in 10 types of organs, including bladder, kidney, lung and pancreas, but was not detected in normal lymph nodes or leukocytes. The data indicated evidence of UP II expression in various types of normal tissues by RT-nested PCR analysis. UP II mRNA was detected in 2 of 11 (18.2%) peripheral blood samples from lung cancer patients with no metastasis, and in 5 of 12 (41.7%) peripheral blood samples of lung cancer patients with metastasis. UP II was also detected in 6 of 16 (37.5%) peripheral blood samples of patients with pancreatic cancer. The data are particularly important in that the molecular detection of micrometastasis in the blood by means of UP II mRNA identification is feasible for UP II-positive neoplasms, including lung and pancreatic cancers.

Novel alternatively spliced variant with a deletion of 52 BP in exon 6 of the progesterone receptor gene is observed frequently in breast cancer tissues.

The human progesterone receptor (PR) is a ligand-activated nuclear transcription factor that mediates progesterone action in target tissues. We found a novel alternatively spliced variant (ASV) of the PR mRNA in breast cancer tissues. The deleted transcript was characterized by an out-of-frame deletion of 52 bp in exon 6 (PR delta6/2 ASV). The PR delta6/2 ASV mRNA results in a partial defect in the region of the ligand-binding domain of the hormone receptor, where conserved residues are missing from the core of the protein. To clarify the clinical significance of the PR delta6/2 ASV, we investigated the expression of this ASV in noncancerous and cancerous tissues from patients with breast cancer using RT-PCR. The novel PR delta6/2 mRNA was detected in 24 of 39 (61.5%) cancerous tissues and in 3 of 39 (7.7%) noncancerous tissues from patients with breast cancer. PR delta6/2 ASV mRNA was expressed more frequently in breast cancer tissues than in noncancerous tissues (p < 0.0001), which suggests a possible relationship between the expression of PR delta6/2 and breast cancer. Our observations may provide a novel strategy for the genetic diagnosis of breast cancer.

Differential alternative splicing expressions of thymidylate synthase isoforms.

We identified two novel deletion variants of the thymidylate synthase transcript in gastric cell lines. Sequence analyses indicate that none of these variants results in introduction of a premature stop-codon or a frame shifts. In 39 gastric cancer samples, both the full-length and one-deletion variant messages were detected in cancerous as well as non-cancerous tissues. However, another isoform was found in only seven of 39 cancerous tissues. Our results provide important information to assist more detailed studies on the regulation of thymidylate synthase activity.

Expression of a novel splicing variant deleting exons 4 and 6 of the progesterone receptor gene is a rare event in breast cancer.

We identified a novel alternatively spliced isoform of PR mRNA in breast cancer tissues. The deleted transcript was characterized by an out-of-frame deletion of 437 bp, corresponding to the complete loss of exons 4 and 6 (PR delta4+6 ASV). PR delta4+6 ASV will result in a partial defect in the region of the ligand-binding domain of hormone receptors, suggesting that the conserved residues are missing from the core of the protein. In the limited number of samples studied, a novel PR delta4+6 mRNA was detected in 1 of 45 (2.2%) non-cancerous tissues of patients with breast cancer, in 5 of 45 (11.1%) cancerous tissues of patients with breast cancer. Loss of both exons 4 and 6 will be induced by incomplete splicing and/or repair mechanism. Further studies are necessary to establish the biological significance of this alternative splicing. The expressions of ASVs that induced the mimic PR transcripts need to be considered when designing strategies for regulation analysis of the PR gene.

Detection of circulating prostate tumor cells: alternative spliced variant of PSM induced false-positive result.

RT-nested PCR has been introduced as a highly specific and sensitive assay method to detect the prostate-specific membrane antigen (PSM) mRNA in peripheral blood. However, appreciable percentages of false-positive cases have been reported. Additionally, primer sets reported previously could not discriminate between PSM and PSM', an alternatively spliced variant, mRNA. These isoforms can be produced from a single gene. Switches in alternative splicing patterns are often controlled with strict cell-type or developmental-stage specificity. Therefore, it is most important to discriminate between PSM mRNA and PSM' mRNA. Using our highly specific primer sets, PSM mRNA was detected in 3 of 24 peripheral blood samples of normal male volunteers (12.5%) and was not detected in peripheral blood of 11 normal female volunteers. PSM' mRNA was detected in 5 of 24 peripheral blood samples of normal male volunteers (20.8%) and in 4 of 11 of normal female volunteers (36.4%). PSM' mRNA induced false-positive results, it is important for genetic diagnosis of prostate cancer to discriminate between PSM and PSM' using our primer sets with high specificity. The advances in the uniquely designed primer sets may allow researchers to detect a real PSM mRNA without PSM' mRNA.

Expression of molecular marker genes in various types of normal tissue: implication for detection of micrometastases.

Many researchers have investigated the expressions of candidates for a suitable reverse transcription nested polymerase chain reaction (RT-PCR) marker. But typically biomarkers often have false-positive results. We assessed whether epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA) and prostate-specific membrane antigen (PSM) could be detected in 28 different types of normal human sources. Using RT-nested PCR assay, EGFR mRNA was also detected in various types of normal tissue, including pancreas, prostate and uterus. CEA was detected in various types of normal tissue, including prostate, uterus, bladder and spleen. PSM mRNA was also detected in various types of normal tissue, including kidney, liver, skeletal muscle, spleen, bladder and ovary. We report here that the expression of these biomarkers in normal cells might have induced false-positives, and that further enhancement of sensitivity might compromise specificity. Conversely, these biomarkers can be utilized for attempts to define micrometastases in various types of tumors whose cells express these tissue-specific genes.

ARHI/NOEY2 inactivation may be important in breast tumor pathogenesis.

Allelic loss frequently occurs on the short arm of chromosome 1 in human breast carcinoma, suggesting that the ARHI/NOEY2 gene, an imprinted putative tumor suppressor gene, is involved in the pathogenesis of the tumor entity. To clarify the clinical importance of ARHI/NOEY2 mRNA in breast cancer, we studied whether ARHI/NOEY2 inactivation might contribute to tumors arising in the breast. An ARHI/NOEY2 message was detected by real-time PCR analysis in all noncancerous breast tissues, but was not detected in 2 of 26 breast cancer tissue samples. In 10 of 26 breast cancer tissue samples ARHI/NOEY2 mRNA was substantially reduced. ARHI/NOEY2 expression was lost or markedly reduced in 12 of 26 (46.15%) breast cancer tissue samples. In summary, we conclude that decreased ARHI/NOEY2 mRNA expression may play an important role in the pathogenesis of breast cancer.