Sex-specific expression of gastrin-releasing peptide receptor: relationship to smoking history and risk of lung cancer.

BACKGROUND: Activation of gastrin-releasing peptide receptor (GRPR) in human airways has been associated with a proliferative response of bronchial cells to gastrin-releasing peptide and with long-term tobacco use. The GRPR gene is located on the X chromosome and escapes X-chromosome inactivation, which occurs in females. Increasing evidence demonstrates that women are more susceptible than men to tobacco carcinogenesis. We hypothesized that the susceptibility of women to the effects of tobacco may be associated with airway expression of GRPR. METHODS: We analyzed GRPR messenger RNA (mRNA) expression in lung tissues and cultured airway cells from 78 individuals (40 males and 38 females) and in lung fibroblasts exposed to nicotine in vitro. Nicotinic acetylcholine receptors in airway cells were assayed by use of radioactively labeled nicotine and nicotine antagonists. A polymorphism in exon 2 of the GRPR gene was used to detect allele-specific GRPR mRNA expression in some individuals. Statistical tests were two-sided. RESULTS: GRPR mRNA expression was detected in airway cells and tissues of more female than male nonsmokers (55% versus 0%) and short-term smokers (1-25 pack-years [pack-years = number of packs of cigarettes smoked per day multiplied by the number of years of smoking]) (75% versus 20%) (P =.018 for nonsmoking and short-term smoking females versus nonsmoking and short-term smoking males). Female smokers exhibited expression of GRPR mRNA at a lower mean pack-year exposure than male smokers (37.4 pack-years versus 56.3 pack-years; P =.037). Lung fibroblasts and bronchial epithelial cells exhibited high-affinity, saturable nicotinic acetylcholine-binding sites. Expression of GRPR mRNA in lung fibroblasts was elevated following exposure to nicotine. CONCLUSIONS: Our results suggest that the GRPR gene is expressed more frequently in women than in men in the absence of smoking and that expression of this gene is activated earlier in women in response to tobacco exposure. The presence of two expressed copies of the GRPR gene in females may be a factor in the increased susceptibility of women to tobacco-induced lung cancer.

Evidence for autocrine actions of neuromedin B and gastrin-releasing peptide in non-small cell lung cancer.

Gastrin-releasing peptide (GRP), a member of the bombesin family of peptides, has been shown to have mitogenic activity in small cell lung carcinoma (SCLC), and to be produced by SCLC in an autocrine fashion. In this report, we demonstrate that both GRP and another member of the bombesin family of peptides, neuromedin B (NMB), are also autocrine growth factors for non-small cell lung carcinoma (NSCLC). Using the reverse transcription-polymerase chain reaction (RT-PCR), we have detected mRNA for the neuromedin B receptor (NMBR) in all 14 of the NSCLC cell lines examined. GRP receptor (GRPR) mRNA was also expressed in the majority of NSCLC cell lines (nine of 14). By immunoblotting using SDS-PAGE gradient gels fixed in trichloroacetic acid, GRP and NMB were found in fractions of culture medium that had been purified by high pressure liquid chromatography (HPLC) from NSCLC cell lines. NMB was detected in the conditioned medium of seven of nine cell lines and GRP in seven of nine cell lines; both peptides were produced in six cell lines. In four of the cell lines where both peptides were produced, the relative amount of NMB secreted into the medium was 7-15 times that of GRP; in the other two cases, the relative amounts of GRP and NMB were equivalent. Cultured human bronchial epithelial (HBE) cells expressed the GRPR and NMBR but did not produce either peptide. A subline of A549 cells that was adapted to grow in serum-free and growth factor-free conditions, termed A549-R(0), secreted both bombesin-like peptides (BLPs) into the culture medium. Using either a colony-forming assay or a BrDU incorporation assay, both NMB and GRP were found to be mitogens for three NSCLC cell lines that express mRNA for BLP receptors and secrete BLPs, regardless of which peptide and/or receptor subtype was detected. The monoclonal antibody 2A11, which preferentially recognizes GRP, was able to block the in vitro proliferative response to GRP in the BrDU incorporation assay, and partially blocked the response to NMB. The 2A11 antibody could only partially block the in vivo growth of cell lines that showed proliferative responses to BLPs. 2A11 antibody was more effective against the 239T cell line, which secreted a low amount of GRP into the medium (0.6 nM), compared to the 201T cell line, which secreted a higher amount of both GRP and NMB (4.2 nM and 36.6 nM, respectively). These results suggest that both NMB and GRP are autocrine growth factors for NSCLC, but that the production of NMB and expression of the NMBR may be more prominent than the production of GRP and expression of the GRP receptor. If BLP ligand-receptor systems are to be targeted therapeutically in NSCLC, it will be necessary to inhibit both NMB and GRP.

Detection of micrometastases in histologically negative lymph nodes in esophageal cancer.

BACKGROUND: New molecular techniques may identify micrometastases in histologically negative lymph nodes and have an impact on the staging of esophageal cancer. We investigated the role of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay to identify micrometastases in esophageal cancer. METHODS: The RT-PCR assay to detect carcinoembryonic antigen (CEA) messenger ribonucleic acid (mRNA) was performed on lymph nodes from patients with esophageal cancer and benign esophageal disorders. The presence of CEA mRNA in lymph nodes was considered evidence of metastases. RESULTS: Histopathologic study revealed metastases in 50 (41%) of 123 lymph nodes from 30 patients with esophageal cancer. All histologically positive lymph nodes contained CEA mRNA by RT-PCR. Of 73 histologically negative lymph nodes, 36 (49%) contained CEA mRNA, a significant increase compared with the histopathologic diagnosis (p < 0.001). Lymph nodes in patients with benign disease contained no CEA mRNA. In 10 patients, histologic stage was NO. Five of them were also negative by RT-PCR, and all are alive with only one recurrence. In the remaining 5 patients, RT-PCR was positive for occult lymph node metastases; 2 have died of disease, and 1 is alive with recurrent disease. CONCLUSIONS: In patients with esophageal cancer, RT-PCR detects more lymph node metastases than does histopathology. Initial follow-up suggests a positive RT-PCR with negative histologic findings may have poor prognostic implications. Further studies will be needed to confirm any clinical implications.

Detection of occult lymph node metastases in esophageal cancer by minimally invasive staging combined with molecular diagnostic techniques.

BACKGROUND AND OBJECTIVES: Lymph node metastases are the most important prognostic factor in patients with esophageal cancer. Histologic examination misses micrometastases in up to 20% of lymph nodes evaluated. In addition, non-invasive imaging modalities are not sensitive enough to detect small lymph nodes metastases. The objective of this study was to investigate the use of reverse transcriptase-polymerase chain reaction (RT-PCR) of messenger RNA (mRNA) for carcinoembryonic antigen (CEA) to increase the detection of micrometastases in lymph nodes from patients with esophageal cancer. METHODS: RT-PCR of CEA mRNA was performed in lymph nodes from patients with malignant and benign esophageal disease. Each specimen was examined histopathologically and by RT-PCR and the results were compared. RESULTS: Metastases were present in 29 of 60 (48%) lymph nodes sample by minimally invasive staging from 13 patients with esophageal cancer when examined histopathologically. RT-PCR identified nodal metastases in 46 of these 60 (77%) samples. RT-PCR detected CEA mRNA in all 29 histologically positive samples and in 17 histologically negative lymph nodes. All lymph nodes from patients with benign disease (n = 15) were negative both histopathologically and by RT-PCR. The stage of two patients was reclassified based on the RT-PCR results, which identified lymph node spread undetected histopathologically. Both of these patients developed recurrent disease after resection of the primary tumor. CONCLUSIONS: RT-PCR is more sensitive than histologic examination in the detection of lymph node metastases in esophageal cancer and can lead to diagnosis of a more advanced stage in some patients. The combination of minimally invasive surgical techniques in combination with new molecular diagnostic techniques may improve our ability to stage cancer patients.

Trinucleotide repeat length variation in the human ribosomal protein L14 gene (RPL14): localization to 3p21.3 and loss of heterozygosity in lung and oral cancers.

Chromosome 3p is consistently deleted in lung cancer, oral squamous cell carcinoma, and renal cell carcinoma, and is believed to contain several tumor suppressor genes. We have shown a role for chromosome 3 in tumor suppression by microcell-mediated chromosome transfer experiments. We have isolated a gene that is located at 3p21.3 within the smallest region of deletion overlap in lung tumors and is the human homolog of the ribosomal protein L14 gene (RPL14). The RPL14 sequence contains a highly polymorphic trinucleotide repeat array which encodes a variable-length polyalanine tract. Genotype analysis of RPL14 shows that this locus is 68% heterozygous in the normal population, compared with 25% in non-small cell lung cancer (NSCLC) cell lines (p = 0.008). Cell cultures derived from normal bronchial epithelium show a 65% level of heterozygosity, reflecting that of the normal population. Squamous cell carcinoma of the head and neck (SCCHN), which has the same risk factors as lung cancer and is hypothesized to have a similar etiology, demonstrates 54% loss of heterozygosity at the RNA level, suggesting that transcriptional loss may be a primary mechanism of RPL14 alteration in SCCHN. In addition, RPL14 shows significant differences in allele frequency distribution in ethnically-defined populations, making this sequence a useful marker for the study of ethnicity-adjusted lung cancer risk.