Activity of the ubiquitously expressed Na؉ -H ؉ exchanger subtype NHE1 is stimulated upon activation of receptor tyrosine kinases and G protein-coupled receptors. The intracellular signaling pathways mediating receptor regulation of the exchanger, however, are poorly understood. Using transient expression of dominant interfering and constitutively active alleles in CCL39 fibroblasts, we determined that the GTPases Ha-Ras and G␣13 stimulate NHE1 through distinct signaling cascades. Exchange activity stimulated by constitutively active RasV12 occurs through a Raf1-and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase kinase (MEK)-dependent mechanism. Constitutively active G␣13QL, recently shown to stimulate the Jun kinase cascade, activates NHE1 through a Cdc42-and MEK kinase (MEKK1)-dependent mechanism that is independent of Rac1. Constitutively active Rac1V12 does stimulate NHE1 through a MEKK1-dependent mechanism, but dominant interfering Rac1N17 does not inhibit G␣13QL-mediated or constitutively active Cdc42V12-mediated stimulation of the exchanger. Conversely, Cdc42N17 does not inhibit Rac1V12 activation of NHE1, suggesting that Rac1 and Cdc42 independently regulate a MEKK1-dependent activation of the exchanger. Rapid (<10 min) stimulation of NHE1 with a G␣13/G␣ z chimera also was inhibited by a kinase-inactive MEKK. G␣13QL, but not RasV12, also stimulates NHE1 through a RhoA-dependent pathway that is independent of MEKK, and microinjection of mutationally active G␣13 results in a Rho phenotype of increased stress fiber formation. These findings indicate a new target for Rho-like proteins: the regulation of H ؉ exchange and intracellular pH. Our findings also suggest that a MEKK cascade diverges to regulate effectors other than transcription factors.The ubiquitously expressed Na ϩ -H ϩ exchanger subtype NHE1 1 plays a major role in intracellular pH (pH i ) homeostasis and in cell volume regulation (1). NHE1 activity is stimulated by hormones, cytokines, and growth factors, resulting in an increase in pH i . Hyperosmotic shock (2) and cell adhesion (3) also activate NHE1. Increases in NHE1 activity are associated with increased cell proliferation (4, 5), differentiation (6, 7), and neoplastic transformation (8 -10). Receptor (11, 12), but not osmotic (2), activation of NHE1 is associated with increased phosphorylation of the exchanger on serine residues, suggesting kinase-dependent regulatory mechanisms. Although activation of protein kinase C stimulates NHE1, growth factors and vasoactive agents can stimulate the exchanger independently of this kinase. Mutational activation of three GTPases, Ha-Ras (8, 9), G␣ q (13-15), and G␣13 (13-15), stimulates NHE1 activity. Of these GTPases, only G␣ q activates the exchanger through a protein kinase C-dependent mechanism (14).2 The downstream signaling events mediating Ha-Ras and G␣13 stimulation of NHE1 have not been identified. Ha-Ras and G␣13 regulate two parallel MAP kinase signaling cascades, and these cascades include serine/threonine p...

Matrix metalloproteinase-2 (MMP-2) plays important roles in cancer development and aggression. Our previous studies revealed a strong association between the MMP-2 ؊1306C/T polymorphism and risk of several cancers. A novel ؊735C/T polymorphism in MMP-2 promoter has been identified but the function is undefined. This study examined our hypothesis that these two polymorphisms might have functional relevance and impact on risk of esophageal squamous cell carcinoma in the context of haplotype. Genotypes and haplotypes were analyzed in 527 cases and 777 controls and odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The function of the polymorphisms was examined by electrophoretic mobility shift assays, luciferase gene expression assays, and reverse transcriptase-PCR analyses. It was found that the ؊735C3 T transition disrupts an Sp1 site and displays a lower promoter activity. The C -1306 -C -735 haplotype had 7-fold increased luciferase expression and 3.7-fold increased MMP-2 mRNA levels in esophageal tissues compared with the T -1306 -T -735 haplotype. A case-control analysis revealed a 1.52-fold (95% CI ‫؍‬ 1.17-1.96) or 1.30-fold (95% CI ‫؍‬ 1.04 -1.63) excess risk of developing esophageal squamous cell carcinoma for the ؊1306CC or ؊735CC genotype carriers compared with noncarriers, respectively. A greater association was observed between elevated risk of developing esophageal squamous cell carcinoma and C -1306 or C -735 allele containing haplotypes, with the risk being highest for the C -1306 -C -735 haplotype compared with the T -1306 -T -735 haplotype (OR ‫؍‬ 6.53; 95% CI ‫؍‬ 2.78 -15.33). The C -1306 -C -735 haplotype was also associated with increased risk for distant metastasis of esophageal squamous cell carcinoma (OR ‫؍‬ 3.34; 95% CI ‫؍‬ 1.16 -9.63). These findings suggest that the C -1306 -C -735 haplotype in the MMP-2 promoter contributes to risk of the occurrence and metastasis of esophageal squamous cell carcinoma by increasing expression of MMP-2.

Variation in DNA repair capacity, which is believed to be largely determined by genetic traits, is linked to risk of certain cancers. The Asp312Asn and Lys751Gln polymorphisms in the xeroderma pigmentosum complementary group D (XPD) gene may alter DNA repair capacity. We thus examined the hypothesis that these 2 XPD polymorphisms are associated with risk of lung cancer via a large hospital-based, case-control study among Chinese. The study subjects consisted of 1,006 patients with primary lung cancer and 1,020 age-and sex-matched population controls. XPD genotypes were determined using PCR-RFLP techniques, and the associations between genotypes and risk of lung cancer were estimated by odds ratios (ORs) and their 95% confidence intervals (CIs) calculated by unconditional logistic regression. The DNA repair system plays an important role in protecting against mutagenesis and carcinogenesis. It has been documented that the defect in DNA repair causes several hereditary cancer syndromes 1 and the development of some common sporadic cancers may also associated with reduced DNA repair capacity. [2][3][4] Accumulating evidence indicates that variation in DNA repair capacity is likely to be largely determined by genetic traits. The defect of DNA repair often results from gene mutations. However, single nucleotide polymorphisms (SNPs), when located within the coding and/or regulating regions of the gene, can also spoil DNA repair capacity due to the amino acid substitution or diminished protein expression. XPD (xeroderma pigmentosum complementary group D), an important DNA repair protein, encodes an evolutionarily conserved ATP-dependent helicase that participates in both nucleotide excision repair and basal transcription as part of the transcription factor TFIIH. 5 Mutations at different sites in XPD that destroy XPD protein function cause 3 severe syndromes: Cockayne's syndrome, trichotiodystrophy and xeroderma pigmentosum, which exhibits a Ͼ1,000-fold incidence of sun-induced skin cancer and elevated risk of internal cancers. 6,7 Several SNPs have also been identified in the XPD locus. Among them, a G-to-A transition in codon 312 of exon 10 results in an Asp3 Asn substitution in an evolutionarily conserved region, and another transversion, A-to-C in codon 751 of exon 23, produces a Lys3 Gln substitution. 8 The 2 sites are reported to be in linkage disequilibrium and appear to have phenotypic significance, although contradictory results exist regarding which allele is associated with impaired DNA repair capacity. 4,9 -14 Since XDP is one of the important components in the nucleotide excision repair (NER) and NER is the most flexible pathway that has the ability to remove a broad range of DNA damage such as BPDE-DNA adducts induced by benzo(a)pyrene, 15,16 a major constituent of tobacco smoking, the impact of these 2 genetic variations in XPD on risk of cancer in addition to skin cancer has been attracting research interest. Some case-control studies have been conducted in different ethnic populations to investigate ...

Matrix metalloproteinase-2 (MMP-2) plays important roles in cancer initiation and progression. Our previous studies revealed that the -1306C-->T and -735C-->T polymorphisms in MMP2 promoter significantly influence transcriptional activity and their genotypes and haplotypes are associated with susceptibility to several cancers. This case-control study examined the contribution of these two polymorphisms to the risk of developing lung cancer. MMP2 genotypes and haplotypes were determined in 770 cases and 777 controls and the associations with risk of lung cancer were estimated by logistic regression. We observed a 2-fold [odds ratio (OR), 2.12; 95% confidence interval (CI), 1.64-2.72] or 1.6-fold (OR, 1.57; 95% CI, 1.27-1.95) excess risk of developing lung cancer for the -1306CC or -735CC genotype carriers compared with non-carriers, respectively. A greater risk of lung cancer was associated with the C(-1306)-C(-735) haplotype (OR, 5.01; 95% CI, 2.57-9.78) compared with the T(-1306)-T(-735) haplotype, suggesting a synergic effect of these two polymorphisms. Furthermore, a greater than additive joint effect of the polymorphisms and smoking increased an even higher risk of lung cancer. The OR for smokers with the C(-1306)-C(-735) haplotype was 6.24 (95% CI, 4.51-8.64), which was significantly higher than that (OR, 4.10; 95% CI, 2.89-5.81) of smokers with the T(-1306) or T(-735)-containing haplotypes (P < 0.001). These results are consistent with our previous findings and further support the hypothesis that gain-of-function of MMP2 resulting from genetic polymorphisms plays an important role in human carcinogenesis.

Aurora-A/BTAK/STK15, involved in regulating centrosomes and chromosome segregation, is overexpressed in human breast carcinoma and other cancers. The Phe31-->Ile polymorphism in Aurora A alters the kinase function, with the Ile31 variant being preferentially amplified and associated with degree of aneuploidy in human tumors. We have previously shown that the Phe31Ile polymorphism is associated with the occurrence and advanced disease status of esophageal cancer. This case-control study examined the contribution of this polymorphism to susceptibility to development and progression of breast cancer. Aurora A genotypes were determined in 520 patients with breast carcinoma, 191 patients with benign breast diseases (BBD) and 520 controls. It was found that the Aurora A Ile/Ile genotype was significantly associated with increased risk of breast carcinoma occurrence [odds ratio (OR) 1.66; 95% confidence interval (95% CI) 1.29-2.12] compared with the Phe/Phe or Phe/Ile genotype. The increased risk for BBD and breast carcinoma related to the Ile/Ile genotype was more pronounced in younger subjects. Moreover, we found that patients carrying the Ile/Ile genotype tended to have ER-carcinomas (OR 2.56; 95% CI 1.24-5.26). No significant association was observed between the polymorphism and metastasis and disease stage of the cancer. These findings suggest that the Phe31Ile polymorphism in Aurora A may be a genetic modifier for developing breast carcinoma.