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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.

Ultraviolet radiation (UVR) may protect against prostate cancer via a mechanism involving vitamin D. Thus, the vitamin D receptor (VDR) gene is a susceptibility candidate, though published data are discrepant. We studied the association of prostate cancer risk with five VDR single nucleotide polymorphisms (SNPs): G/A(1229) (SNP 1), A/G(3944) (SNP 2), T/C(30875) (SNP 3), C/T(48200) (SNP 4) and C/T(65013) (SNP 5), in 430 cancer and 310 benign prostatic hypertrophy (BPH) patients. The SNP 2 GG genotype frequency was lower in cancer than BPH patients (odds ratio = 0.63, 95% CI = 0.41-0.98, p = 0.039). SNPs 1 and 2, and SNPs 4 and 5, were in linkage disequilibrium. Two copies of haplotypes comprising SNPs 1-2, G-G (odds ratio = 0.63, p = 0.039), SNPs 2-3 G-C (odds ratio = 0.45, p = 0.008) and SNPs 1-2-3 G-G-C (odds ratio = 0.44, p = 0.006), but not SNPs 1-3, G-C (odds ratio = 0.81, p = 0.34), were associated with reduced risk (reference, no copies of the haplotypes). These associations were observed after stratification of subjects by extent of UVR exposure. These data show that SNP 2 GG genotype mediates prostate cancer risk, complementing studies reporting this allele is protective in malignant melanoma pathogenesis. They further suggest that published associations of risk with SNP 1 may result from linkage disequilibrium with SNP 2.

Summary We have examined the association of the CCND1 A/G 870 polymorphism with susceptibility and outcome in 174 German patients with oral SCC (OSCC). The CCND1 G 870 allele frequency was increased in cases (G 870 = 0.65) when compared to controls ( n = 155, G 870 = 0.54) and the distribution of CCND1 genotypes were significantly different ( p = 0.014). Using logistic regression, correcting for age, gender and tobacco consumption, an increased frequency of the CCND1 GG 870 genotype was observed in the OSCC cases ( p = 0.025, OR 3.37, 95% CI 1.61–9.80). No significant associations were observed between CCND1 A/G 870 and tumour histological factors. Our data suggests that the CCND1 GG 870 genotype is associated with increased susceptibility to OSCC. The involvement of cyclin D1 polymorphism in mechanisms of SCC development may differ in the different sub-sites of the head and neck.

Splicing of human cyclin D1 (CCND1) mRNA producing transcripts a and b is modulated by a common polymorphism (A --G) located in a conserved splice donor region at nucleotide 870. CCND1 A/G(870) genotype is associated with tumour progression and clinical outcome in a variety of cancers. Although in vitro expression of cyclin D1 transcript a (CCND1(tra)) has been widely investigated, few studies have examined the expression of CCND1 transcript b (CCND1(trb)). We have studied the effects of inducible expression of human CCND1(trb) in comparison with human CCND1(tra) in a mouse fibroblast knock-out for cyclin D1 (MEF(Cyl-1-/-)). Inducible expression was in stable clones isolated from MEF(Cyl-1-/-) transfectants. Induction of CCND1(tra) produced a 36-kDa protein, which led to a significant increase in the proportion of cells in S-phase, as detected by BrdU incorporation after 32 hr, compared to non-induced cells (p = 0.012). Clones induced to express CCND1(tra) exhibited a significantly increased ability to grow in serum depleted (2% FCS) medium compared to non-induced clones (p = 0.0004). Induced expression of CCND1(trb) in MEF(Cyl-1-/-) transfectants produced a 31-kDa protein and resulted in no significant difference in DNA synthesis, neither did the cells acquire the ability to grow in serum-depleted conditions compared to non-induced cells. Induction of CCND1(trb) significantly enhanced the ability of MEF(Cyl-1-/-) transfectants to form colonies in soft agar, (average 30-fold increase) compared to non-induced clones or those induced to express CCND1(tra). Our data supports the emerging view that CCND1 alternate transcripts encode proteins with differing independent biological functions. We suggest that CCND1(tra) encodes a protein involved in regulating mitogen responsive, anchorage-dependent G(1) progression, whereas CCND1(trb) modulates the ability of the cell to grow in an anchorage-independent manner.

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.

Cognitive impairment occurs in 45—65% of multiple sclerosis (MS) patients. The cannabinoid system may potentially be neuroprotective in MS. We examined the relationship between polymorphisms of the CNR1 gene and neuropsychological outcome in MS using a test and confirmatory sample of patients. One hundred and ninety-four MS patients were assessed over five key areas of neuropsychological function, which are most commonly impaired in MS. The first 97 patients formed the test sample. A further confirmatory sample of 97 patients was used to test association found in the test sample. The schedule included: Wisconsin card sorting test 64 version, Rey auditory verbal learning task immediate and delayed scores, controlled oral word association task, judgement of line orientation and symbol digit modalities task. Three single nucleotide polymorphisms (SNPs) were typed within the CNR1 gene. For the overall neuropsychological assessment score we used a multiple linear regression model with selected covariates to show that subjects with the AA genotype of the SNP RS1049353 were more impaired (mean -2.47, SD 5.75, P = 0.008, Bonferroni corrected P = 0.024) than the other subjects (mean 0.24, SD 4.24). This was not confirmed when the association was retested in the confirmatory sample. No associations were identified between these CNR1 variants and cognitive impairment in MS. Multiple Sclerosis 2008; 14: 177—182. http://msj.sagepub.com

Genes implicated in tumor evolution and progression, including those in apoptotic pathways, are associated with methylation-associated gene silencing in different tumor types. By exploiting differential methylation we recently isolated a novel pituitary tumor derived apoptosis gene (PTAG) that augments drug-induced apoptosis. The importance of PTAG was determined in other tumor types, and these studies show that the majority of primary colorectal tumors fail to express the PTAG gene, indicating an important role for PTAG in colorectal tumorigenesis. The effects of expression of PTAG were examined through stable transfection of the colorectal cell lines HCT116 and SW480. Expression of PTAG, per se, had no discernible effects on cell viability or cell kinetics. In contrast to these findings, in cells subject to drug challenges that engaged either a death-receptor mediated or mitochondrial pathway, all of the experiments indicated a role for PTAG in the intrinsic pathway of apoptosis. Loss of PTAG therefore contributes to a blunted apoptotic response and is likely to predispose cells toward malignant transformation and resistance to chemotherapeutic interventions.

Cancer is a world-wide health problem associated with an increasing death rate. The mechanisms of how normal cells transform into cancer cells are not fully understood. Intensive investigations have been undertaken to identify genes whose unregulated expression are involved in this process. In this study, we have grown, on collagen gel, adherent mouse embryo fibroblasts (MEFs) knocked out for Cyl-1 (MEFCyl1-/-) which have been transfected with the human proto-oncogene cyclin D1 (CCND1) under the control of an inducible expression system. CCND1 expression can be regulated in the fibroblasts via the presence of an inducer, isopropyl β-D-Thiogalactopyranoside (IPTG). In the absence of IPTG, CCND1 expression is silenced. The migration ability of the resultant cells on the collagen gel has been monitored by complementary optical techniques: the conventional light microscopy; optical coherence tomography and Fourier Transform Infrared Microspcopic Spectroscopy (FTIR) using Synchrotron beam source. It is found that the cells expressing CCND1 exhibited cell invasion morphology and had different matrix compositions near the cell layer in comparison to the cells not expressing CCND1. The results from this study are consistent with published findings that expression of CCND1 has oncogenic potential and is involved in cell invasion in vitro. Application of complementary optical techniques proves to be an efficient way obtaining morphological and composition information of cell invasion.

Glutathione S-transferase (GST) enzymes catalyse the detoxification of by-products of reactive oxygen species and are thus important in cellular defence mechanisms. The GSTs are polymorphic with allelic variants encoding isoforms with functional differences. GST polymorphism has been associated with susceptibility and clinical outcome in patients with cancer. In this retrospective cohort, we have investigated associations between common GSTM1, GSTM3 and GSTP1 polymorphisms with factors known to influence clinical out-come and patient survival in colorectal cancer. Significant linkage disequilibrium was demonstrated between GSTM1 and GSTM3 alleles (P< or =0.001). We identified no significant associations between the GSTP1(Ile105Val105) polymorphism and any clinical outcome parameters or patient survival. However significant associations were demonstrated with mu class GSTs. Those patients who were GSTM1 null presented less frequently with poorly-differentiated tumours (P=0.038). Furthermore, patients who were GSTM3 AA were less likely to present with advanced stage tumours (T-stage, P=0.036 and Dukes' classifications, P=0.012) or distant metastases (P=0.017) when examined alone. Upon further examination of the effect of linkage disequilibrium, we found that, in GSTM1 null individuals, GSTM3 AA (compared with other GSTM3 genotypes combined) had longer disease-free survival (HR=0.54, 95% CI 0.30-0.98, P=0.044). Thus, the GSTM3 AA genotype is associated with improved prognosis especially in those with GSTM1 null. Our findings suggest that the GST mu gene cluster mediates tumour characteristics and survival in patients with colorectal cancer.

Glutathione S-transferase (GST) enzymes catalyse the detoxification of by-products of reactive oxygen species and are thus important in cellular defence mechanisms. The GSTs are polymorphic with allelic variants encoding isoforms with functional differences. GST polymorphism has been associated with susceptibility and clinical outcome in patients with cancer. In this retrospective cohort, we have investigated associations between common GSTM1, GSTM3 and GSTP1 polymorphisms with factors known to influence clinical out-come and patient survival in colorectal cancer. Significant linkage disequilibrium was demonstrated between GSTM1 and GSTM3 alleles (Por =0.001). We identified no significant associations between the GSTP1(Ile105Val105) polymorphism and any clinical outcome parameters or patient survival. However significant associations were demonstrated with mu class GSTs. Those patients who were GSTM1 null presented less frequently with poorly-differentiated tumours (P=0.038). Furthermore, patients who were GSTM3 AA were less likely to present with advanced stage tumours (T-stage, P=0.036 and Dukes' classifications, P=0.012) or distant metastases (P=0.017) when examined alone. Upon further examination of the effect of linkage disequilibrium, we found that, in GSTM1 null individuals, GSTM3 AA (compared with other GSTM3 genotypes combined) had longer disease-free survival (HR=0.54, 95% CI 0.30-0.98, P=0.044). Thus, the GSTM3 AA genotype is associated with improved prognosis especially in those with GSTM1 null. Our findings suggest that the GST mu gene cluster mediates tumour characteristics and survival in patients with colorectal cancer.

To determine mechanisms for pituitary neoplasia we used methylation-sensitive arbitrarily primedPCR 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 M) 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 zVAD-fmk. In addition, in time course experiments, direct measurement of active caspases by fluorochrome-labeled inhibition of caspases, showed an augmented increase (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. (Molecular Endocrinology 18: 1827–1839, 2004)