We have previously reported that the cyclin D1 (CCND1) GG 870 genotype was associated with poorly differentiated tumors and reduced disease-free interval in patients with squamous cell carcinoma of the head and neck (SCCHN). We have now examined the association of this and a second CCND1 polymorphism with gene expression and outcome in SCCHN patients. Analysis of a CCND1 G/C 1722 polymorphism revealed that CCND1 CC 1722 genotype was associated with poorly differentiated tumors [P ؍ 0.005; odds ratio (OR), 5.7; 95% CI, 1.7 to 19.2), and reduced disease-free interval (P ؍ 0.003; Hazard Ratio (HR), 7.3; 95% CI, 1.1 to 27.2.) independently from the influence of CCND1 GG 870 genotype. Patients whose tumors were negative for cyclin D1 were associated with reduced disease-free interval (P ؍ 0.028; HR, 4.1; 95% CI, 1.4 to 14.2). Although G/C 1722 genotypes were not associated with expression, we found a significant trend between reduced expression of cyclin D1 in patients with the CCND1 GG 870 genotype (P ؍ 0.04). Splicing of CCND1 mRNA in head and neck tissues was modulated by CCND1 A/G 870 alleles, thus CCND1 transcript a was spliced equally from CCND1 A 870 and G 870 alleles, whereas CCND1 transcript b was spliced mainly from the CCND1 A 870 allele. Our analysis has also identified differences in cyclin D1 genotype and protein expression and the pathogenesis of SCCHN in males and females. Thus, CCND1 CC 1722 genotype was more common in female patients (P ؍ 0.019; OR, 3.3; 95% CI, 1.3 to 10) and cyclin D1 expression was more frequent (chi-square 1 , 3.96; P ؍ 0.046) and at higher levels (P ؍ 0.004) in tumors from female patients. In summary, our data show that the two CCND1 polymorphic sites are independently associated with tumor biology and clinical outcome. CCND1 A/G 870 alleles affect gene expression in head and neck tissues. We also provide preliminary evidence that the molecular genetics of SCCHN development may be influenced by patient gender. (Am J Pathol 2001, 159:1917-1924)
To determine mechanisms for pituitary neoplasia we used methylation-sensitive arbitrarily primed-PCR to isolate novel genes that are differentially methylated relative to normal pituitary. We report the isolation of a novel differentially methylated chromosome 22 CpG island-associated gene (C22orf3). Sodium bisulfite sequencing of pooled tumor cohorts, used in the isolation of this gene, showed that only a proportion of the adenomas within the pools were methylated; however, expression analysis by quantitative RT-PCR of individual adenoma irrespective of subtype showed the majority (30 of 38; 79%) failed to express this gene relative to normal pituitary. Sodium bisulfite sequencing of individual adenomas showed that 6 of 30 (20%) that failed to express pituitary tumor apoptosis gene (PTAG) were methylated; however, genetic change as determined by loss of heterozygosity and sequence analysis was not apparent in the remaining tumors that failed to express this gene. In those cases where the CpG island of these genes was methylated it was invariably associated with loss of transcript expression. Enforced expression of C22orf3 in AtT20 cells had no measurable effects on cell proliferation or viability; however, in response to bromocriptine challenge (10-40 microm) cells expressing this gene showed a significantly augmented apoptotic response as determined by both acridine orange staining and TUNEL labeling. The apoptotic response to bromocriptine challenge was inhibited in coincubation experiments with the general caspase inhibitor z-VAD-fmk. In addition, in time course experiments, direct measurement of active caspases by fluorochrome-labeled inhibition of caspases, showed an augmented increase (approximately 2.4 fold) in active caspases in response to bromocriptine challenge in cells expressing C22orf3 relative to those harboring an empty vector control. The pituitary tumor derivation and its role in apoptosis of this gene led us to assign the acronym PTAG to this gene and its protein product. The ability of cells, showing reduced expression of PTAG, to evade or show a blunted apoptotic response may underlie oncogenic transformation in both the pituitary and other tumor types.
Expression of the glutathione S-transferase, GSTP1, is associated with phase 1 detoxification of the products of oxidative stress. Recently, GSTP1 expression has been implicated in the regulation of cell proliferation and apoptosis through direct interaction with the c-Jun N-terminal kinase, (JNK). GSTP1 is polymorphic and allelic variants have been associated with disease susceptibility and clinical outcome. However, the influence of GSTP1 alleles on proliferation and apoptosis has not been studied previously. To investigate this, we have examined the effects of inducible expression of wild-type GSTP1*A and mutant GSTP1*C haplotypes on cell proliferation and apoptosis in NIH3T3 fibroblasts. Cells expressing GSTP1*A displayed increased doubling times and a delayed G1-S phase transition compared with cells expressing GSTP1*C. Both GSTP1*A and GSTP1*C haplotypes protected cells from undergoing apoptosis when exposed to oxidative stress. However, analysis of JNK status revealed that only GSTP1*C expression led to a reduction in JNK activity compared with GSTP1*A-expressing cells and non-induced cells. We further examined the effect of GSTP1 alleles on colony-forming efficiency (CFE) in soft agar following exposure to oxidative stress and found that GSTP1*A-expressing clones had increased CFE compared with non-induced and GSTP1*C-expressing clones. Our data suggest that GSTP1 alleles have differential effects on proliferation and apoptosis; GSTP1*A reduces cellular proliferation and protects against apoptosis through a JNK-independent mechanism. In contrast, GSTP1*C does not influence cellular proliferation but protects cells from apoptosis through JNK-mediated mechanisms.
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