Histonedeacetylase 1 mRNA has elevated expression in clinical specimen of bladder cancer.

OBJECTIVE: HDACs are among transcriptional regulatory elements that regulate key features of proliferation and differentiation in all cell types including cancerous. They may also interfere in such stages of cancer development as migration, invasion, multi-drug resistance and angiogenesis. Proven information about HDAC1 role in development of bladder cancer is limited only to cell lines in vitro. The lack of a comprehensive clinical in vivo study led us to evaluate HDAC1 expression in human clinical specimens. METHODS: We analyzed a large group of bladder cancer patients. The presence of hHDAC1 mRNAs were tracked using specific HDAC1 primers in cancer samples and the quantity of HDAC1 transcripts were quantified using real time qPCR method and was compared to those of normal bladder samples from healthy patients. RESULTS: HDAC1 mRNA expression was significantly elevated in Bladder cancer specimens. To our knowledge, this result is the first, showing an elevation in vivo in HDAC1 mRNA levels in clinically cancerous tissue of patients with bladder cancer. CONCLUSIONS: We conclude that hHDAC1 overexpression might be implicated in bladder cancer tumorigenesis and that the over-expressed HDAC1 mRNA might be a potential diagnostic marker and, a target for treatment of bladder cancer using HDACi-drugs in future (Tab. 2, Fig. 2, Ref. 30).

Sperm mitochondrial DNA deletion in Iranian infertiles with asthenozoospermia.

Asthenozoospermia is an important cause of male infertility. The mutations in sperm mitochondrial DNA (mtDNA) result in either functionless or malfunctioning some proteins, subsequently affecting sperm motility leading to asthenozoospermia. The purpose of this study was to investigate sperm mtDNA 4,977-bp deletion in infertile men with low sperm motility/immotile spermatozoa compared to healthy subjects with high sperm motility. Semen samples of 256 asthenozoospermic infertiles and 200 controls from northern Iran were collected. After extraction of spermatozoa total DNA, Gap-polymerase chain reaction (Gap-PCR) was performed. The deletion was observed in 85.93% of patients with asthenozoospermia compared with 14% in controls [OR = 37.5397, 95% confidence interval = 12.937-108.9276, p < .0001]. It is concluded that there is a strong association between sperm mtDNA 4,977-bp deletion and asthenozoospermia-induced infertility in the population examined. Large-scale mtDNA deletions in spermatozoa may induce bioenergetic disorders. Nevertheless, to validate our results broader research may be needed.

Genetic Variants miR-126, miR-146a, miR-196a2, and miR-499 in Polycystic Ovary Syndrome.

Introduction: Alterations in certain microRNAs (miRNAs) and their target genes have reported in polycystic ovary syndrome (PCOS) and other disease of the female reproductive system, and so may be potential biomarkers. We hypothesised alterations in the prevalence of four miRNAs single nucleotide polymorphism (SNP) variants miR-126 rs4636297, miR-146a rs2910164, miR-196a2 rs11614913, and miR-499 rs3746444 in women with PCOS in comparison to healthy controls. Methods: SNPs in the four miRNAs were determined in 385 patients and 385 controls by standard RT-PCR techniques. Results: SNPs in miR-126 and miR-246a were significant linked with PCOS under the allelic, dominant, co-dominant, and recessive models (all p ≤ 0.01). The SNP in miR-499 was linked to PCOS in allelic (T, p = 0.002), dominant (p = 0.035) and recessive (p = 0.003) models. The SNPs -196a was significant linked to PCOS only in the recessive model (p = 0.037). Combining these SNPs in miR-499, mi146a, miR-196a and miR-126 respectively into allele haplotypes found highly significant odds ratios (95% CI) of 0.40 (0.29-0.54) (p < 0.001) for the C-G-C-G haplotype, and 0.46 (0.30-0.70) (p = 0.002) for the C-C-C-A haplotype (p = 0.002) for PCOS. Conclusion: Single SNPs and haplotype combinations in certain SNPs in miR-126, miR-146a, miR-196a2 and miR-499 are strongly linked to PCOS, and so may be useful predictors of this condition.

Association analysis of KISS1 polymorphisms and haplotypes with polycystic ovary syndrome.

INTRODUCTION: KISS1 play an essential role in human reproductive functions by regulating the hypothalamic-pituitary-gonadal axis. Loss-of-function mutations in this gene have been frequently identified in patients with different reproductive disorders. We hypothesised links between KISS1 polymorphisms and polycystic ovary syndrome (PCOS). MATERIALS AND METHODS: In order to find links between KISS1 polymorphisms rs4889 C > G, rs12998 G > A, and rs35431622 A > G with PCOS, 770 blood samples were obtained from 385 control and 385 PCOS women. DNA was extracted, and genotyped for KISS1 variants by PCR. RESULTS: rs12998 G > A was linked to PCOS in dominant (p < 0.001), recessive (p < 0.001), co-dominant (p < 0.001), and allelic models (p < 0.001). In addition, rs4889 C > G was linked in recessive, dominant, co-dominant, and allelic models (p < 0.001). rs35431622 A > G was not linked to PCOS. Further analysis indicated that C-G-G haplotype was more common and G-A-G haplotype was less prevalent in cases compared with controls. CONCLUSION: KISS1 variants rs12998 G > A and rs4889 C > G may be linked to the pathophysiology of PCOS.

MicroRNA variants in endometriosis and its severity.

Background: MicroRNAs (miRNAs) are naturally occurring posttranscriptional regulatory molecules that potentially play a role in endometriotic lesion development.Method: We evaluated the prevalence of miRNAs variants miR-146a rs2910164, miR-149 rs2292832, miR-196a2 rs11614913, and miR-499 rs3746444 in endometriosis in 260 cases and 260 controls. DNA was extracted and genotyping of the SNPs was carried out by PCR.Results: There was a significant association of rs2910164 and rs2292832 with increased rates of endometriosis under the dominant (p < 0.001), recessive (p < 0.05), co-dominant (p < 0.001), and allelic model (p < 0.001). Also, rs3746444 showed a borderline association with endometriosis under the recessive model (p = 0.05); however, rs11614913 was not linked to endometriosis. Further analysis indicated the significant association of miR-146a rs2910164 polymorphism genotypes (GG, GC, and CC) and miR-149 rs2292832 genotypes (CC and CT) with endometriosis severity in patients (p < 0.001). Additionally, haplotype frequency in cases compared to controls and Linkage disequilibrium (LD) between the mentioned SNPs was appraised.Conclusion: MiR-146a, miR-149 and miR-499 may have a role in the pathogenesis of endometriosis.

Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span.

Normal somatic cells have a finite life span [1] and lose telomeric DNA, present at the ends of chromosomes, each time they divide as a function of age in vivo or in culture [2-4]. In contrast, many cancer cells and cell lines established from tumours maintain their telomere length by activation of an RNA-protein complex called telomerase, an enzyme originally discovered in Tetrahymena [5], that synthesizes telomeric repeats [6-8]. These findings have led to the formation of the 'telomere hypothesis', which proposes that critical shortening of telomeric DNA due to the end-replication problem [9] is the signal for the initiation of cellular senescence [10,11]. In yeast, the EST2 gene product, the catalytic subunit of telomerase, is essential for telomere maintenance in vivo [12-14]. The recent cloning of the cDNA encoding the catalytic subunit of human telomerase (hTERT) [15,16] makes it possible to test the telomere hypothesis. In this study, we expressed hTERT in normal human diploid fibroblasts, which lack telomerase activity, to determine whether telomerase activity could be reconstituted leading to extension of replicative life span. Our results show that retroviral-mediated expression of hTERT resulted in functional telomerase activity in normal aging human cells. Moreover, reconstitution of telomerase activity in vivo led to an increase in the length of telomeric DNA and to extension of cellular life span. These findings provide direct evidence in support of the telomere hypothesis, indicating that telomere length is one factor that can determine the replicative life span of human cells.

Analysis of genomic integrity and p53-dependent G1 checkpoint in telomerase-induced extended-life-span human fibroblasts.

Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279-282, 1998; A. G. Bodnar et al., Science 279:349-352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21(Waf1/Cip1/Sdi1). TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.

A new mathematical modeling for pure parsimony haplotyping problem.

Pure parsimony haplotyping (PPH) problem is important in bioinformatics because rational haplotyping inference plays important roles in analysis of genetic data, mapping complex genetic diseases such as Alzheimer's disease, heart disorders and etc. Haplotypes and genotypes are m-length sequences. Although several integer programing models have already been presented for PPH problem, its NP-hardness characteristic resulted in ineffectiveness of those models facing the real instances especially instances with many heterozygous sites. In this paper, we assign a corresponding number to each haplotype and genotype and based on those numbers, we set a mixed integer programing model. Using numbers, instead of sequences, would lead to less complexity of the new model in comparison with previous models in a way that there are neither constraints nor variables corresponding to heterozygous nucleotide sites in it. Experimental results approve the efficiency of the new model in producing better solution in comparison to two state-of-the art haplotyping approaches.

Alternative pathways for the extension of cellular life span: inactivation of p53/pRb and expression of telomerase.

Telomere shortening may be one of several factors that contribute to the onset of senescence in human cells. The p53 and pRb pathways are involved in the regulation of cell cycle progression from G1 into S phase and inactivation of these pathways leads to extension of life span. Short dysfunctional telomeres may be perceived as damaged DNA and may activate these pathways, leading to prolonged arrest in G1, typical of cells in senescence. Inactivation of the p53 and pRb pathways, however, does not lead to cell immortalization. Cells that overcome senescence and have an extended life span continue to lose telomeric DNA and subsequently enter a second phase of growth arrest termed 'crisis'. Forced expression of telomerase in human cells leads to the elongation of telomeres and immortalization. The development of human cancer is frequently associated with the inactivation of the pRb and p53 pathways, attesting to the importance of senescence in restricting the tumor-forming ability of human cells. Cancer cells must also maintain telomere length and, in the majority of cases, this is associated with expression of telomerase activity.

From telomere loss to p53 induction and activation of a DNA-damage pathway at senescence: the telomere loss/DNA damage model of cell aging.

In the cold winter of 1966 Aleksay Olovnikov, a theoretical biologist at the Academy of Sciences in Moscow, was waiting in the subway station where he was hit by the idea that the ends of linear chromosomes can't be replicated fully during each round of replication. In a theoretical paper (Olovnikov, 1971) he proposed that in somatic cells the ends of the chromosomes are not fully replicated during DNA synthesis, resulting in the shortening of linear DNA molecules with each cell division, and that this may be the cause of cell cycle arrest in senescent cells. Almost two decades after this proposal, Calvin Harley and co-workers found that telomeres, the physical ends of human chromosomes, shorten as a function of age in human cells in vitro and in vivo. The telomere hypothesis proposes that critically short telomeres may act as a mitotic clock to signal the cell cycle arrest at senescence (Harley, 1991). Here, we extend the telomere hypothesis and propose a model that incorporates recent advances in tumor suppressors and cell cycle control with several areas of cell aging. We propose that telomere shortening per se is not the direct signal for cell cycle arrest. It is the consequence of telomere loss, which may lead to generation of ds or ss DNA breaks. These breaks activate a p53 dependent or independent DNA-damage pathway that leads to the induction of a family of inhibitors of cyclin dependent kinases (including p21 and p16) and the eventual G1 block of senescence. In agreement with this hypothesis, we demonstrate that the level of p53 protein increases in near senescent cultures of MDFs. This increase may be responsible for induction of p21 (Noda, 1993) and IGF-Bp3 (Goldstein, 1991).

Ultrasonic biomicroscopy of viable, dead and apoptotic cells.

Ultrasonic imaging is frequently used in medical diagnosis to differentiate normal and tumour tissues. Here we investigate if distinct types of cell death can be discriminated through the use of ultrasound biomicroscopy. By using a well-controlled system in vitro, we demonstrate that this imaging modality can be used to differentiate living cells, dead cells and cells that have died by programmed cell death or apoptosis. The results indicate a greater than twofold ultrasound backscatter signal from apoptotic cells in comparison to viable cells, whereas heat-killed cells exhibit an intermediate level of ultrasound backscatter. The results have potential implications in the study of disease-related biological processes involving apoptosis.

Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming.

AIM: To determine whether transcriptional reprogramming is capable of reversing the developmental aging of normal human somatic cells to an embryonic state. MATERIALS & METHODS: An isogenic system was utilized to facilitate an accurate assessment of the reprogramming of telomere restriction fragment (TRF) length of aged differentiated cells to that of the human embryonic stem (hES) cell line from which they were originally derived. An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2, OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length, telomerase activity and telomere-related gene expression. In addition, we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. RESULTS: We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09-21.1 kb) but markedly shorter TRF lengths (6.4-12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However, iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines. In order to verify these results in an isogenic background, we generated six iPS cell clones from the hES-derived cell line EN13. These iPS cell clones showed initial telomere lengths comparable to the parental EN13 cells, had telomerase activity, expressed embryonic stem cell markers and had a telomere-related transcriptome similar to hES cells. Subsequent culture of five out of six lines generally showed telomere shortening to lengths similar to that observed in the widely distributed iPS lines. However, the clone EH3, with relatively high levels of telomerase activity, progressively increased TRF length over 60 days of serial culture back to that of the parental hES cell line. CONCLUSION: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.

Critical telomere shortening regulated by the ataxia-telangiectasia gene acts as a DNA damage signal leading to activation of p53 protein and limited life-span of human diploid fibroblasts. A review.

Somatic cells undergo a limited number of doublings in culture and enter an irreversible block in the G1 and G2/M phase of the cell cycle termed "senescence". Telomere shortening presumably as a consequence of the end-replication problem has been proposed to act as a mitotic clock eventually leading to cellular senescence. Several models have been proposed to explain how telomere shortening can lead to cellular senescence. We proposed previously that telomere shortening may eventually lead to formation of dicentric chromosomes which on subsequent breakage activate a DNA damage response pathway involving the p53 protein. Hence we proposed that the telomere shortening signal is perceived by the cell as DNA damage. Recently we have obtained experimental evidence that the p53 protein is activated posttranslationally in human fibroblasts which undergo telomere shortening and subsequent senescence in culture. In this paper we also show that the increased activity of p53 protein coincides with formation of dicentric chromosomes and senescence. Also, we have previously found that an increase in the level of the down stream target of p53 protein, p21WAF1/SD11/C1P1, is dependent on both p53 and p300 proteins. We have also shown that fibroblasts obtained from individuals with Ataxia Telangiectasia lose telomeric DNA at an accelerated rate, activate p53 protein, and undergo premature senescence in culture. These results suggest that the ataxia-telangiectasia gene (ATM) and p53 are involved in surveillance and regulation of telomeric DNA. Once a critical length of telomeric DNA is reached. ATM and p53 sense and relay this signal to the cell cycle leading to senescence.

Characteristics of drug abusers in an adolescent in-patient psychiatric facility.

Characteristics related to the abuse of drugs in 143 adolescents who had been institutionalized in an inpatient psychiatric facility were investigated. A factor analysis generated the following clusters: (1) multiple drug usage, (2) male narcotics procurement, (3) drug history, (4) prognostic index, (5) family drug usage, (6) entrepreneurial procurement of drugs, and (7) social procurement of drugs. Results also indicated that our patients are coming to us at increasingly younger ages and that patients admitted in 1969 appear more seriously involved in drug abuse than those admitted before or after this time.

Deproteination of nucleic acids by filtration through a hydrophobic membrane.

To remove or inactivate an enzyme from DNA in multistep procedures in molecular biology, it is often necessary to phenol extract the solution, followed by chloroform extraction and ethanol precipitation. In addition to being time-consuming and hazardous, there can be significant loss of DNA with this procedure, especially when small volumes or amounts of DNA are being used. We have found that filtering analytical reaction mixtures through a hydrophobic membrane specifically to remove protein is a rapid alternative to phenol extraction. Within broad limits commonly encountered in molecular biology, filtration through a polyvinylidene difluoride membrane quantitatively removes a variety of enzymes without significant loss of double-stranded nucleic acid.