Mutation analysis of the PTEN/MMAC1 gene in lung cancer

We studied PTEN/MMAC1, a newly discovered candidate tumor suppressor gene at 10q23.3, for mutations in lung cancer. One hundred and thirty-six lung cancer cell line DNAs (66 small cell lung cancers, SCLC, 61 non-small cell lung cancers, NSCLC, four mesotheliomas, five extrapulmonary small cell cancers) were analysed for PTEN/MMAC1 homozygous deletions and five (8%) SCLC lines showed homozygous deletions interrupting the PTEN/MMAC1 gene. Using single stranded conformation polymorphism (SSCP) analysis, we screened the PTEN/MMAC1 open reading frame of 53 lung cancer cell line cDNAs for point mutations and found that 3/35 SCLCs and 3/18 NSCLCs contained homozygous amino acid sequence altering mutations. Northern blot analysis revealed that expression of the PTEN/MMAC1 gene was considerably lower in all the tumor cell lines with point mutations while no expression was detected for cell lines with PTEN/MMAC1 homozygous deletions. Mutation analysis of 22 uncultured, microdissected, primary SCLC tumors and metastases showed two silent mutations, and two apparent homozygous deletions. We also discovered a processed pseudogene (PTEN2) which has 98.5% nt identity to PTEN/MMAC1, that needs to be accounted for in cDNA mutation analysis. Our findings suggest that genetic abnormalities of the PTEN/MMAC1 gene are only involved in a relatively small subset of lung cancers.     

Genomic organization and mutation analysis of Hel-N1 in lung cancers with chromosome 9p21 deletions

Allelic loss of chromosome 9p21 is common in small cell lung cancer (SCLC), but inactivation of the tumor suppressor gene CDKN2a is rare, implying the existence of another target gene at 9p21. A recent deletion mapping study of chromosome 9p has also identified a site of deletion in non-small cell lung cancer (NSCLC) centered around D9S126. The Hel-N1 (human elav-like neuronal protein 1) gene encodes a neural-specific RNA binding protein that is expressed in SCLC. We have mapped this potentially important gene in lung tumorigenesis to within 100 kb of the D9S126 marker at chromosome band 9p21 by using homozygously deleted tumor cell lines and fluorescence in situ hybridization to normal metaphase spreads. Hel-N1 is, therefore, a candidate target suppressor gene in both SCLC and NSCLC. We have determined the genomic organization and intron/exon boundaries of Hel-N1 and have screened the entire coding region for mutations by sequencing 14 primary SCLCs and cell lines and 21 primary NSCLCs preselected for localized 9p21 deletion or monosomy of chromosome 9. A homozygous deletion including Hel-N1 and CDKN2a was found in a SCLC cell line, and a single-base polymorphism in exon 2 of Hel-N1 was observed in eight tumors. No somatic mutations of Hel-N1 were found in this panel of lung tumors. Hel-N1 does not appear to be a primary inactivation target of 9p21 deletion in lung cancer.     

Determinants of CPT-11 and SN-38 activities in human lung cancer cells

Irinotecan (CPT-11) is a semisynthetic camptothecin derivative with a broad spectrum of anti-tumour activity. Carboxylesterase (CE) catalyses the conversion of CPT-11 to SN-38 (7-ethyl-10-hydroxycamptothecin), the active form of CPT-11. The antiproliferative effects of CPT-11 and SN-38, CE-activity and topoisomerase I protein expression were investigated in five human small-cell lung cancer (SCLC) cell lines and four human non-small-cell lung cancer (NSCLC) cell lines. Antiproliferative activity, expressed as IC50 values, was determined using the MTT assay. CPT-11 was significantly more active in SCLC than in NSCLC cell lines (P = 0.0036), whereas no significant difference between histological types was observed with SN-38. A significant correlation (r2 = 0.52, P = 0.028) was observed between CE activity and chemosensitivity to CPT-11 but not to SN-38, and significantly higher CE activity was observed in SCLC compared with NSCLC cell lines (P = 0.025). Western blotting experiments showed topoisomerase I protein expressions within a factor of 2, and a granular nuclear staining was detectable in all cell lines by immunocytochemistry of cytospins. No correlation was observed between protein expression and sensitivity to CPT-11 or SN-38. Cellular and medium concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography (HPLC) in one SCLC cell line with high CE activity and high sensitivity to CPT-11, and one NSCLC cell line with low sensitivity to CPT-11 and CE activity. Intracellular concentrations of CPT-11 and SN-38 were higher in the SCLC cell line, and this was associated with an increase in cellular uptake of CPT-11 compared with the medium, and an increased intracellular formation of SN-38. In conclusion, CE activity appears to be associated with higher sensitivity to CPT-11 in human lung cancer cell lines and may partly explain the difference in the in vitro sensitivity to CPT-11 between SCLC and NSCLC cells. The assessment of CE activity in clinical material of lung cancer patients undergoing treatment with CPT-11 may be warranted. However, other mechanisms may influence sensitivity to CPT-11, possibly including drug transport.     

Nerve growth factor abrogates the tumorigenicity of human small cell lung cancer cell lines

Nerve growth factor (NGF) has antiproliferative and differentiating effects on adenomas of neuroendocrine origin. Cell lines derived from small-cell lung carcinoma (SCLC), a very aggressive neuroendocrine tumor, express NGF receptors. The role of NGF in the control of proliferation and progression of this carcinoma, however, has never been investigated. Chronic exposure of NCI-N-592 and GLC8 SCLC cell lines to NGF remarkably inhibited their proliferation rate both in vitro and in vivo, prevented their anchorage-independent clonal growth in soft agar, impaired their invasive capacity in vitro, and abolished their tumorigenic potential in nude mice. The proliferative response of SCLC cell lines to nicotine was also remarkably impaired by in vitro NGF treatment. Furthermore, NGF treatment activates in SCLC cell lines the expression and secretion of NGF. NGF thus reverts SCLC cell lines to a noninvasive, nontumorigenic phenotype that does not respond to nicotine and produces NGF.     

Clinical correlates of bombesin-like peptide receptor subtype expression in human lung cancer cells

The importance of the expression of the autocrine growth system for bombesin-like peptides (BLPS) to the biological behavior of human lung cancer has not been determined. Three BLP receptor subtypes have been identified in human lung and lung cancer cells: gastrin-releasing peptide (GRP) receptor, neuromedin B (NMB) receptor, and bombesin receptor subtype 3 (BRS-3). The goals of this study were: (1) to determine BLP receptor subtype expression by human lung cancer cell lines by RT/PCR; (2) to evaluate possible clinical correlates of characteristics of the patients from whom the cell lines were derived with patterns of BLP receptor expression. Degenerate PCR primers were designed to amplify all known BLP receptors and yielded products from 19/20 small cell lung carcinoma (SCLC) and 12/13 non-small cell lung carcinoma (NSCLC) cell lines. GRP receptor was the most commonly expressed BLP receptor subtype, being detected in 17/20 SCLC and 11/13 NSCLC. Eleven of 20 SCLC expressed NMB receptors, and 5/20 expressed BRS-3, compared with 4/13 and 1/13, respectively, in NSCLC cell lines. Evaluation of the clinical data of the patients from whom the cell lines were derived revealed expected age, sex, smoking history and survival based on histology and stage. Patients from whom cell lines expressed GRP receptor experienced a better survival than those whose cell lines did not (367 +/- 274 days vs. 211 +/- 114 days), but the results were not statistically significant. RT/PCR analysis is a feasible, sensitive and specific means of determining BLP receptor expression in lung cancer cells and may yield prognostic information in patient tissue.     

Steroid-hormone receptors in cell lines and tumor biopsies of human lung cancer

Female gender is a significant independent favorable prognostic factor in lung cancer. To study the possible role of sex hormones in lung cancer, the expression of sex-steroid receptors and the glucocorticoid receptor was investigated in 29 lung-cancer cell lines stemming from small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) by means of immunocytochemistry, ligand-binding assays and RNA expression via polymerase chain reaction. In at least 2 methods of investigation, NSCLC cell lines showed a low expression of estrogen receptor in 6, progesterone receptor in 13 and androgen receptor in 12 out of 17 cases examined; sex-steroid-receptor expression was virtually absent in SCLC cell lines. The glucocorticoid receptor was expressed in all 29 cell lines studied. Additionally, 52 tumor samples from primary lung cancer were investigated for their receptor expression by means of immunohistochemistry. Among patients with primary lung-cancer sex-steroid-receptor expression in tumor biopsies was detected most frequently in female patients (in 69% of 16 cases, vs. 42% of 36 tumors from men) and in patients with adenocarcinoma. Further research will focus on these subgroups. Immunohistology is a feasible method of studying steroid-receptor expression in lung cancer.     

Immortalization of neuroendocrine pinealocytes from transgenic mice by targeted tumorigenesis using the tryptophan hydroxylase promoter

Tryptophan hydroxylase (TPH) is the first enzyme in both serotonin and melatonin biosynthesis in neuroendocrine cells of the pineal gland. The lack of immortalized neuroendocrine pineal cell lines has been a major obstacle to the study of the tissue-specific and circadian regulation of TPH gene expression in the pineal gland. Previously, we demonstrated that a 6.1 kb 5' upstream region of the mouse TPH gene directs the restricted expression of a lacZ reporter gene to the pineal gland and the raphe nuclei of transgenic mice. Therefore, to develop TPH-expressing pineal cell lines we first established transgenic mice carrying a construct consisting of 6.1 kb of 5' flanking region fused to the SV40 T-antigen. These animals developed highly invasive pineal tumors and died at 12-15 weeks of age. The pineal tumors obtained from the transgenic mice were utilized to establish the immortalized pinealocyte-derived cell lines. These cells express two marker enzymes, TPH and serotonin N-acetyltransferase (NAT). In pineal gland TPH and NAT expressions have been known to be regulated during circadian cycle. The two established cell lines therefore promise to be a valuable in vitro model system for the study of the rhythmic nature of the pineal function at molecular level in mammal.