Passive and post-exercise cold-water immersion augments PGC-1α and VEGF expression in human skeletal muscle.

PURPOSE: We tested the hypothesis that both post-exercise and passive cold water immersion (CWI) increases PGC-1α and VEGF mRNA expression in human skeletal muscle. METHOD: Study 1 Nine males completed an intermittent running protocol (8 × 3-min bouts at 90 % [Formula: see text], interspersed with 3-min active recovery (1.5-min at 25 % and 1.5-min at 50 % [Formula: see text]) before undergoing CWI (10 min at 8 °C) or seated rest (CONT) in a counterbalanced, randomised manner. Study 2 Ten males underwent an identical CWI protocol under passive conditions. RESULTS: Study 1 PGC-1α mRNA increased in CONT (~3.4-fold; P < 0.001) and CWI (~5.9-fold; P < 0.001) at 3 h post-exercise with a greater increase observed in CWI (P < 0.001). VEGFtotal mRNA increased after CWI only (~2.4-fold) compared with CONT (~1.1-fold) at 3 h post-exercise (P < 0.01). Study 2 Following CWI, PGC-1α mRNA expression was significantly increased ~1.3-fold (P = 0.001) and 1.4-fold (P = 0.0004) at 3 and 6 h, respectively. Similarly, VEGF165 mRNA was significantly increased in CWI ~1.9-fold (P = 0.03) and 2.2-fold (P = 0.009) at 3 and 6 h post-immersion. CONCLUSIONS: Data confirm post-exercise CWI augments the acute exercise-induced expression of PGC-1α mRNA in human skeletal muscle compared to exercise per se. Additionally CWI per se mediates the activation of PGC-1α and VEGF mRNA expression in human skeletal muscle. Cold water may therefore enhance the adaptive response to acute exercise.

PGC-1α transcriptional response and mitochondrial adaptation to acute exercise is maintained in skeletal muscle of sedentary elderly males.

The aim of the present study was to examine the effects of ageing and training status on (1) markers of skeletal muscle mitochondrial content and (2) the ability to activate the acute signalling pathways associated with regulating exercise-induced mitochondrial biogenesis. Muscle biopsies were obtained from the vastus lateralis muscle of young untrained (24 ± 4 years, n = 6; YU), young trained (22 ± 3 years, n = 6; YT), old untrained (65 ± 6 years, n = 6; OU) and old trained (64 ± 3 years, n = 6; OT) healthy males before and after (3 h and 3 days post-exercise) completion of high-intensity interval cycling exercise. In resting muscle, lifelong training preserved mtDNA, PGC-1α and COXIV protein content such that muscles from OT individuals were comparable to muscles from both YU and YT individuals, whereas lifelong sedentary behaviour reduced such markers of mitochondrial content. Regardless of age or training status, acute exercise induced comparable increases in p38MAPK phosphorylation immediately post-exercise, PGC-1α and COXIV mRNA expression at 3 h post-exercise and COXIV protein at 3 days post-exercise. Data demonstrate that lifelong endurance training preserves skeletal muscle PGC-1α content and that despite the mitochondrial dysfunction typically observed with sedentary ageing, muscles from sedentary elderly individuals retain the capacity to activate the acute signalling pathways associated with regulating the early processes of mitochondrial biogenesis. We consider our data to have immediate translational potential as they highlight the potential therapeutic effects of exercise to induce skeletal muscle mitochondrial biogenesis persist late in adulthood, even after a lifetime of physical inactivity.

DBC1 re-expression alters the expression of multiple components of the plasminogen pathway.

Deleted in bladder cancer 1 (DBC1) is a candidate gene for the bladder tumour suppressor locus at 9q33.1. The function of the gene is currently unknown but a cross-species sequence comparison suggests an important role, as it is highly evolutionarily conserved. Here, we transfected a nonexpressing human bladder cancer cell line with a set of human DBC1 cDNA constructs. The effect on global expression patterns was assessed using cDNA microarrays. The cell clone with the lowest level of DBC1 expression showed induced expression of 26 genes including plasminogen activator inhibitor 2 (SERPINB5; 4.6-fold), heparin-binding EGF-like growth factor precursor (DTR; 4.2-fold), small proline-rich protein 2B (SPRR2B; 3.6-fold), metallothionein 1 isoforms (MT1B/MT1A/MT-1F; from 2.9- to 3.2-fold), tissue-type plasminogen activator precursor (PLAT; 2.8-fold) and urokinase-type plasminogen activator precursor (PLAU; 2.7-fold). In clustering analysis, both PLAT and PLAU clustered with the functionally related urokinase plasminogen activator surface receptor (PLAUR; 1.9-fold). Furthermore, 14 human bladder tumours were analysed by real-time quantitative PCR using gene-specific primers for selected (n=20) genes. The expression levels of SERPINB5, PLAU, PLAUR and MT1 correlated with the DBC1 levels, suggesting previously unknown involvement of DBC1 in the urokinase-plasminogen pathway.

Destabilization of chromosome 9 in transitional cell carcinoma of the urinary bladder.

The most frequent genetic alteration in transitional cell carcinoma of the urinary bladder (TCC) is loss of chromosome 9 which targets CDKN2A on 9p. The targets on 9q are not confirmed. Here, 81 advanced TCC specimens were investigated for loss of heterozygosity (LOH) and homozygous deletions (HD) on chromosome 9q using multiplex analysis of microsatellite markers. 41/81 tumours (51%) showed LOH on 9q, with LOH at all markers in 33 cases. Eight partial losses involved three regions in 9q12, 9q22.3, and 9q33- 9q34. No mutations were identified in the candidate tumour suppressor gene DBCCR1 in three tumours showing restricted LOH at 9q32-33. 22% of the specimens had HD at CDKN2A, but no HD was found on 9q. Two tumours had lost 9p only and five 9q only. 9q LOH was not related to tumour grade or stage and present or absent with equal frequency in recurrent TCC. LOH on 9q correlated with the extent of genome-wide hypomethylation (P < 0.0001) which extended into satellite sequences located in 9q12 juxtacentromeric heterochromatin. While the high frequency of chromosome 9q loss in TCC may reflect destabilization of the chromosome related to hypomethylation of repetitive DNA, the data are compatible with the existence of tumour suppressor genes on this chromosome arm.

Loss of heterozygosity in tumour-adjacent normal tissue of breast and bladder cancer.

Normal tumour-adjacent breast tissue samples from 12 breast cancer patients forming six monozygotic twin pairs were analysed for loss of heterozygosity (LOH) on chromosomes 1, 13 and 17. 7 patients showed LOH at one or more markers. Each of them had a different LOH pattern. Only one twin pair showed LOH at the same locus, but the twins had lost a different allele. Multiple (n=1-13), histologically normal samples were collected from 6 bladder cancer patients and analysed for LOH on chromosomes 3 and 9. On chromosome 9, all 6 patients analysed showed LOH in at least one sample and one marker. Four of them also showed LOH on chromosome 3. Samples surrounding different tumours of a given patient resembled each other. More heterogeneity was seen between the patients, even though they shared some similarities in LOH clustering. The results demonstrate that tumour-adjacent normal tissues already harbour genetic changes typical for tumours. These alterations can reveal the earliest changes leading to tumorigenesis.

The prevalence of loss of heterozygosity in chromosome 3, including FHIT, in bladder cancer, using the fluorescent multiplex polymerase chain reaction.

OBJECTIVE: To determine some of the genetic alterations involved in the pathogenesis and progression of transitional cell carcinoma of the bladder. Materials and methods In a population-based study, freshly frozen tissue was collected from all patients newly diagnosed with urinary bladder cancer in the Stockholm region during 1995-1996. The prevalence of loss of heterozygosity (LOH) was assessed at seven sites on chromosome 3, analysed in 151 patients, using a fluorescent multiplex polymerase chain reaction based on DNA from the tumour and peripheral blood. RESULTS: LOH was detected in 12.1% (at 3q25-26.2) to 22.1% (at 3p11-12) of the informative cases. Relatively frequent LOH was detected at 3p22-24.2 (21.6%), at 3p14.2 within FHIT (21.5%), and at 3p11-12 (22.1%). Of 151 tumours, 72 (47.7%) showed LOH at one or more loci on chromosome 3. LOH on chromosome 3 was weakly associated with tumour grade (P = 0.095), but not with tumour stage (P = 0.701). However, when the frequency of LOH was analysed individually at each site, the prevalence of LOH at 3p11-12 was closely correlated with higher tumour stage (P = 0.011). Replication errors were detected in only four of 151 (2.6%) tumours. Conclusion These findings suggest that the 3p11-12 locus may involve a putative candidate tumour-suppressor gene which might be associated with bladder tumour invasiveness. The FHIT gene locus showed a relatively high frequency of LOH even in Ta tumours.

Multiple regions with allelic loss at chromosome 3 in superficial multifocal bladder tumors.

We have examined six patients with multiple low grade, low stage superficial multifocal bladder tumors with surrounding tissues for loss of heterozygosity (LOH) and microsatellite instability at chromosome 3, totaling 76 samples. The majority (4/5) of the patients had LOH at or close to the fragile histidine triad (FHIT) gene (3p14.2; D3S1300), which is a candidate tumor suppressor gene for many cancer types. One patient showed a consistent LOH with four adjacent markers around FHIT region in all the tumors whereas in the corresponding surrounding tissues the heterozygosity was retained. In addition to the region near FHIT, two other regions had frequent allelic losses - one near the p telomere (3p25-26; D3S3050) and another near the q telomere (3q27; D3S2418). The largest numbers of LOH in the surrounding tissues were found at these regions (3/5 at D3S3050 and 2/5 at D3S2418). The D3S3050 marker is located at 3p26-3p25, near the Von Hippel-Lindau (VHL) tumor suppressor gene locus. LOH that were more random were found at 3q21.3-25.2 (D3S1744) and at 3p12-3p11 (D3S2465). Taken together, at least three regions at chromosome 3p25-26, 3p14.2 and 3q27 seem to have frequent loss of heterozygosity in multifocal superficial bladder tumors. We also performed a phylogenetic-type analysis to find out common changes and the degree of heterogeneity. The overall heterogeneity was low within a given patient: in all cases the majority of the tumor samples arranged in a single branch with a common origin. This point of origin varied from patient to patient, which is compatible with the earlier studies demonstrating the heterogeneity of the single primary bladder tumors. However, the phylogenetic-type analysis suggests that the FHIT region contains often the very first alterations at chromosome 3.

Allelic losses demonstrate monoclonality of multifocal bladder tumors.

The clonality of multifocal bladder tumors has been studied over the years with some controversial results. We have examined 5 patients with 2-11 low-grade superficial multifocal bladder tumors for loss of heterozygosity (LOH) at 87 loci on 9 chromosomes. When LOH was detected at a given marker, the tumors consistently showed deletion of a specific allele, suggesting the monoclonality of the patients' tumors. No allelic imbalancies were detected between heterozygote alleles, and the allelic losses were only slightly biased toward the loss of the shorter alleles as the overall ratio was 0.48 +/- 0.10 (0.50 for nonbiased). We calculated the probabilities for monoclonality using binomial distribution. The use of multiple tumors with multiple microsatellite markers gives high statistical power for the calculation. The combined probabilities for monoclonality varied from 0.984 to (1-4 x 10(-28)). Thus, in most (4/5) cases, the probability for polyclonality was <2 x 10(-16). These results demonstrate that superficial multifocal bladder tumors are most likely of monoclonal origin.

Initiation-development modelling of allelic losses on chromosome 9 in multifocal bladder cancer.

Multiple low-grade, low-stage superficial tumours were analysed for loss of heterozygosity (LOH) on chromosome 9 with 29 markers. Three consensus regions were found, one at 9p (9p21-22) and two at 9q (9q21-31 and 9q32-34). Phylogenetic trees were calculated for each patient using both designated chromosome 9 regions and, separately, using individual microsatellite data. Regional analysis suggested that multiple, equally important regions for bladder tumour initiation exist on chromosome 9. During the development of tumours all regions were eventually affected. The phylogenetic analyses with individual markers were used as molecular clocks to trace the ordering of tumours. The results were compared with the physical locations of the tumours and a hypothetical development model was built. These are novel approaches which, to our knowledge, have not been used before.

Bladder cancer: allelic deletions at and around the retinoblastoma tumor suppressor gene in relation to stage and grade.

Inhibition of the retinoblastoma tumor suppressor gene (RB) is probably important in the pathogenesis of urinary bladder cancer. Little information is available concerning allelic loss on 13q11 to 13q32 and its relation to grade and stage. In a population-based study, freshly frozen tissue was collected from all new cases of urinary bladder cancer in the Stockholm region during 1995-1996. Here we report the occurrence of loss of heterozygosity (LOH) at seven sites in 13q11 to 13q32 as analyzed in 236 cases by a fluorescent multiplex PCR-based on tumor DNA and peripheral blood. For each site, about 30% of the cases were not informative because of homozygosity. Replication errors were detected in 4% (17 cases). LOH was found in 21 (at 13q11-12.1) to 32% (at 13q14.3 in RB) of the informative cases. A correlation was found between the prevalence of LOH at all observed loci and stage and grade, respectively, and it was statistically significant for 13q14.3. LOH at RB was found in Ta as well as grade 1 tumors. Also, a statistically significant correlation was found between the number of loci with LOH at 13q and tumor stage and grade, respectively. Typically an altered RB function is related to the expected clinical course of urinary bladder cancer, but allelic loss including the gene also occurs in low grade and low stage tumors. An altered RB function probably is not necessary for a malignant transformation of urothelial cells. The causal direction of the relation between the quantity of the deleted DNA and tumor aggressiveness is not clear.

Molecular and cellular alterations in tobacco smoke-associated larynx cancer.

Tumours of head and neck belong to the most frequent types of cancer world-wide. In Poland, mortality from larynx cancer among males has been continuously increasing during the last decades up to 8.4 deaths per 100,000 men in 1993, which exceeds epidemiological records from other countries. The aetiology of laryngeal cancer is strongly associated with exposure to carcinogens present in tobacco smoke. The review describes a sequence of molecular and cellular events from carcinogenic exposure, DNA adduct formation, detection of mutations in the p53 gene, loss of heterozygosity (LOH) in chromosomal loci encoding the p53 and p16 genes, and loss of control of the cell cycle. The section concerning DNA adducts includes a discussion of the role of such confounders as exogenous exposure, the age and sex of the subject, and disease progression. The significance of genetic factors as individual risk determinants is discussed in relation to bleomycin-induced chromosome instability and in connection with the occurrence of defects in genes encoding detoxifying enzymes. The question concerning the substantial difference between men and women in larynx cancer morbidity and mortality remains open, even when the significantly higher adduct formation in male DNA compared with female material was taken into account. Preliminary experiments suggest a role of the frequently observed loss of the Y-chromosome.

Mutations in the CDKN2A (p16INK4a) gene in microdissected sporadic primary melanomas.

The role of the CDKN2A (p16INK4a) gene in sporadic primary melanomas has remained unclear due to the inadequate number of mutational studies. In the present study, we analyzed the entire coding region of the CDKN2A gene in microdissected sporadic primary melanomas, for the presence of mutations and polymorphisms, using 2 independent methods of mutation detection, SSCP and CMC. We found 11 intragenic mutations in 8 melanomas out of 31 (26%) and the majority of mutations were located in exon 1, with 2 cases harbouring multiple mutations. Of the mutations detected, 6 were C-to-T transitions, 4 involving CC sites; 2 melanomas showed a novel deletion of one of the two 24-bp repeat units located at the 5' end of exon 1. There was also a high frequency of C-to-G and C-to-T polymorphisms at the nucleotides 540 (frequency of G allele: 0.18) and 580 (frequency of T allele: 0.13) in the 3' untranslated region.

Loss of heterozygosity and microsatellite instability in larynx cancer.

We have examined 48 squamous cell cancers of the larynx for loss of heterozygosity (LOH) and microsatellite instability at chromosome 9p near the p16 tumour suppressor locus, at chromosome 17p at the p53 tumour suppressor locus, and at 17p at another microsatellite locus (D17S520). In p53 LOH was higher (33-45%) than in D17S520 (21%). Near the p16 locus LOH varied from 28-38%. Replication errors were found from at least one locus in 23% of the patients. These data suggest that p53 is an important gene in laryngeal cancer while loci around p16 appear less likely candidates.

Future research directions in the use of biomarkers.

Many DNA adduct studies have been carried out in occupational groups that have been at a risk of cancer based on epidemiological results relating to exposure decades ago. Even new epidemiological publications on cancer cannot accurately address the effective exposures after about 1970. This is one justification for biomarker studies. Another justification is exposures for which epidemiological studies have not been conducted or have provided inadequate results, in spite of suspicions raised by short-term or animal experiments. The modulation of environment carcinogenesis by host polymorphism in genes for xenobiotic metabolizing and DNA repair enzymes is currently under extensive investigation. The studies relating phenotype/genotype to cancer are presently extended to various end points that may be related to cancer such as DNA adducts and cytogenetic damage. Adjustment for a metabolic phenotype or genotype may also increase the precision in the measurement. Mutations in oncogenes and tumor suppressor genes may give clues to the etiology of cancer.

Separation of transforming amino acid-substituting mutations in codons 12, 13 and 61 the N-ras gene by constant denaturant capillary electrophoresis (CDCE).

We used high fidelity PCR and constant denaturant capillary electrophoresis (CDCE) [Khrapko et al. (1994) Nucleic Acids Res., 22, 364-369] to separate wild type and different mutant N-ras exon 1 and 2 sequences. The set of plasmids containing N-ras cDNA, wild type or mutant sequences representing all transforming amino acid-substituting single base pair changes in codons 12, 13 (exon 1) and 61 (exon 2), were amplified using Pfu polymerase in a limited cycle polymerase chain reaction. One of the primers used for the amplification of each exon included a 40 nucleotide GC rich sequence that created high and low melting domains. The amplified fragments 151 bp (exon 1) and 150 bp (exon 2) were run on the CDCE with the 'denaturant zone' temperature of the capillary corresponding to the melting temperature of 111 bp (exon 1) and 110 bp (exon 2) low melting domains. The separation was achieved between wild type and mutant sequences as homoduplexes in 15 out of 19 cases, as a single base substitution alters the electrophoretic mobility of a partially melted double stranded fragment. The denaturation and reannealing of wild type and mutant fragments together created wild type/mutant heteroduplexes. All the heteroduplexes were well resolved from wild type homoduplex. In the current form mutant sequences were detected at a frequency of 10(-3) in the presence of wild type. This study has resulted in obtaining electrophoretic spectrum of different N-ras mutants on CDCE as homoduplexes as well as heteroduplexes.

Post-translational modifications in mitotic yeast cells.

We have recently shown that secretion of invertase is not inhibited in the yeast Saccharomyces cerevisiae during mitosis, but continues, as during interphase. This is in contrast with the mammalian cell, where membrane traffic stops at the onset of prometaphase. Here we extend our findings by showing that the bulk of the cell surface glycoproteins and mannans, as well as the yeast pheromone alpha-factor, traverse the secretory pathway during mitosis. We show that the mitotic cells are able to carry out several types of post-translational modification of secretory proteins. (a) The secretory protein invertase was oligomerized and extensively glycosylated, (b) the N-glycan cores of bulk-cell surface mannans were extended with outer chains, (c) some N-glycans were phosphorylated, (d) the protein-bound O-glycans were extended up to tetramannosides, (e) prepro-ka-factor was proteolytically processed to alpha-factor molecules. We conclude that the secretory pathway in yeast remains fully functional throughout the cell cycle.