[Show abstract][Hide abstract] ABSTRACT: Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR). The nuclear inclusions consisting of the mutant AR protein are characteristic and combine with many components of ubiquitin-proteasome and molecular chaperone pathways, raising the possibility that misfolding and altered degradation of mutant AR may be involved in the pathogenesis. We have reported that the overexpression of heat shock protein (HSP) chaperones reduces mutant AR aggregation and cell death in a neuronal cell model (Kobayashi et al., 2000). To determine whether increasing the expression level of chaperone improves the phenotype in a mouse model, we cross-bred SBMA transgenic mice with mice overexpressing the inducible form of human HSP70. We demonstrated that high expression of HSP70 markedly ameliorated the motor function of the SBMA model mice. In double-transgenic mice, the nuclear-localized mutant AR protein, particularly that of the large complex form, was significantly reduced. Monomeric mutant AR was also reduced in amount by HSP70 overexpression, suggesting the enhanced degradation of mutant AR. These findings suggest that HSP70 overexpression ameliorates SBMA phenotypes in mice by reducing nuclear-localized mutant AR, probably caused by enhanced mutant AR degradation. Our study may provide the basis for the development of an HSP70-related therapy for SBMA and other polyQ diseases.
Journal of Neuroscience 04/2003; 23(6):2203-11. · 6.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dj2 is a member of the DnaJ family of proteins, which regulate the chaperoning function of the hsp70s. We isolated a monkey cDNA dj2 clone corresponding to the large mRNA species encoded by the gene. This mRNA differs from the small mRNA produced by the same gene in that it contains a long 3' untranslated region. Both messages were found to be equally stable and to produce the same protein, which is susceptible to farnesylation. Studies in mouse tissues and various cell lines revealed that these messages and their products are differentially expressed. Surprisingly, we found that only the nonfarnesylated form of dj2 is capable of translocating to the cell nucleus, especially after heat shock. Finally, based on protein interaction studies, our results indicate that dj2 is a specific partner for hsc70 and not for hsp70.
European Journal of Biochemistry 04/2002; 269(5):1553-60. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We used non-denaturing gradient analysis of cell extracts before and after heat treatment of the cells and showed that hsp70 and hsc70 aggregate in vivo in a temperature-dependent fashion. Their aggregation profiles were found to be clearly distinguishable and sensitive to ATP depletion. Pore exclusion limit electrophoresis showed that these two proteins are mainly found in autoaggregated forms including dimers, trimers and oligomers. The addition of denatured luciferase to the cell extracts reversed the aggregation of both proteins towards their non-aggregated forms. Immunoprecipitation and Western-blot analysis showed that the non-aggregated form is the only one bound to denatured luciferase. Our results suggest that aggregated hsp70 and hsc70 represent predominantly self-associated molecules unable to exert chaperone activity. The cochaperone hsp40 was also found to be aggregated and, on addition of denatured luciferase, its aggregation was reversed to a non-aggregated state. Immunoprecipitation analysis indicated that hsp40 forms a complex with the non-aggregated form of hsc70 and denatured luciferase. These results confirm previous in vitro studies and support the suggestion that in vivo cytosolic hsp70 and hsc70 exist mainly in an oligomer–monomer equilibrium which is dependent on the environmental temperature, the levels of ATP and the presence of denatured proteins.
European Journal of Biochemistry. 12/2001; 259(1‐2):505 - 512.
[Show abstract][Hide abstract] ABSTRACT: SV40 belongs to a group of DNA tumor viruses which induce the expression of the 70 Kd heat shock proteins, but the meaning of this induction remains unclear. Investigating the role of hsc70 in the SV40 life cycle, we found that the protein translocates to the nucleus late in infection of permissive CV1 cells, in contrast to infected nonpermissive BALB/3T3 and NIH/3T3 cells in which hsc70 remains cytoplasmic. Moreover, the pattern of hsc70 nuclear staining was diffused and clearly distinguishable from that observed after heat shock. In addition hsc70 late in infection coimmunoprecipitated with the viral capsid protein VP1, suggesting a role in the process of viral packaging. Interactions of hsc70 with the early viral oncoprotein T antigen were observed only in nonpermissive cells, indicating that the binding of the above proteins is specific to cells that do not support viral propagation. Finally, treatment of permissive CV1 cells with interferon gamma, a known antiviral cytokine, resulted in hsc70 binding to T antigen. Our results suggest that the role of hsc70 in the process of SV40 infection is directly related to the ability of the host cells to support viral propagation and is clearly different between permissive and nonpermissive cell lines.
Cell Stress and Chaperones 05/2000; 5(2):132-8. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Indomethacin, a potent anti-inflammatory drug, activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1), but this is insufficient to elevate heat shock gene expression. However, indomethacin pretreatment leads to a complete heat shock response at temperatures that are by themselves insufficient. Here, we showed that the heat-induced loss of enzymatic activity of a nuclear or a cytoplasmic luciferase expressed in murine cells was enhanced when cells had been pretreated with indomethacin. Additionally, in these cells the 70-kDa constitutive heat shock protein exhibited an enhanced aggregation in the presence of indomethacin. Similarly an increase in the aggregation of beta-galactosidase was observed. These data suggest that indomethacin at moderate temperatures accelerates the presence of denatured proteins in the cell, thus lowering the temperature threshold for a heat shock response.
Cell Stress and Chaperones 02/2000; 5(1):8-13. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The establishment of additional powerful prognostic markers in breast cancer patients is of unquestionable importance given that breast cancer is characterized by morphologic, biologic and genetic heterogeneity. In the present study we analyzed 8 primary invasive breast carcinomas by comparative genomic hybridization (CGH) in order to find and map the DNA copy number changes occurring in these tumors. Furthermore, in order to evaluate the potential prognostic significance, we compared these genetic changes with other histo- and immunopathologic prognostic variables, such as tumor type, tumor grade, lymph node status, estrogen receptors content and c-erbB-2 oncoprotein expression. All the studied cases showed a wide variety of gains and losses of chromosomal regions or arms distributed among 16 chromosomes with an average number of 6.12 aberrations per case. Although several genetic changes appeared to be common, none was unique or consistent in all the studied cases. The most consistent regions of gain were on 1q, 20q and 8q while the most common regions of loss on 3p and 6q. Accumulation of chromosomal changes were more frequently found in high grade ductal breast carcinomas with overexpression of c-erbB-2 oncoprotein in both lymph node-negative and lymph node-positive patients, whose tumors were positive for estrogen receptors. If any of these genetic changes identified by CGH in breast cancer patients carry prognostic information, regardless of stage or other factors predictive of biologic behavior, further investigation is needed.
Journal of experimental & clinical cancer research: CR 10/1999; 18(3):357-61. · 1.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied five groups of women with ovarian dysfunction for the CGG expansion in FMR1 and a (TA)n polymorphism in the estrogen receptor gene: a) poor responders to ovarian stimulation as part of in vitro fertilization (n = 13); b) women with familial premature ovarian failure (POF) (n = 7); c) sporadic cases with POF (n = 16); d) FRAXA premutation carriers with POF (n = 7); and e) FRAXA premutation carriers without POF (n = 9). FRAXA premutation was found in one woman with familial POF. A significant association of familial POF and FRAXA premutation carriers with POF having low copy of the (TA)n polymorphism as compared to controls was observed. Our preliminary data suggest a potential role of the estrogen receptor in POF, and it may influence the variable age of menopause of the FRAXA premutation carriers.
American Journal of Medical Genetics 06/1999; 84(3):306-8.
[Show abstract][Hide abstract] ABSTRACT: This study presents the first large, population-based molecular investigation of the fragile X (FRAXA) and FRAXE mental retardation syndromes in the Hellenic populations of Greece and Cyprus. The aims of this population screening were to determine the prevalence of FRAXA and FRAXE syndromes among idiopathic mentally retarded (IMR) individuals, to estimate the incidence in the general population, and to investigate the molecular mechanism of instability and expansion of the FMR1-repeat. Ten FRAXA patients were identified to have either the full mutation (eight) or premutation (two) from a Hellenic population of 866 unrelated IMR individuals (611 males and 255 females, age range 3-25 years). No FRAXE patients were identified among the 611 IMR males. The incidence of FRAXA in the Hellenic population of Cyprus is estimated at 1 in 4,246 males. The repeat sites from the FMR1 and FMR2 alleles were accurately determined and showed similar distribution and frequencies with other population studies. The analysis of AGG interspersion within the FMR1-repeat in normal males revealed long, pure CGG repeats within the "gray zone" as well as variation within the 3' end showing polarity of instability. This finding supports the hypothesis that the AGG interspersion and the length of the pure repeat are major factors in determining allele stability. Analysis of FRAXAC1, DXS548, and FRAXAC2 identified particular alleles and haplotypes to have a significant association with either gray zone alleles or alleles >15 pure CGG repeats. We hypothesize that this subgroup of alleles and haplotypes are associated with long pure CGGs (>15 CGG) or 35 repeats and, having shared an evolutionary past, would have the tendency to expand.
American Journal of Medical Genetics 05/1999; 84(3):184-90.
[Show abstract][Hide abstract] ABSTRACT: We used non-denaturing gradient analysis of cell extracts before and after heat treatment of the cells and showed that hsp70 and hsc70 aggregate in vivo in a temperature-dependent fashion. Their aggregation profiles were found to be clearly distinguishable and sensitive to ATP depletion. Pore exclusion limit electrophoresis showed that these two proteins are mainly found in autoaggregated forms including dimers, trimers and oligomers. The addition of denatured luciferase to the cell extracts reversed the aggregation of both proteins towards their non-aggregated forms. Immunoprecipitation and Western-blot analysis showed that the non-aggregated form is the only one bound to denatured luciferase. Our results suggest that aggregated hsp70 and hsc70 represent predominantly self-associated molecules unable to exert chaperone activity. The cochaperone hsp40 was also found to be aggregated and, on addition of denatured luciferase, its aggregation was reversed to a non-aggregated state. Immunoprecipitation analysis indicated that hsp40 forms a complex with the non-aggregated form of hsc70 and denatured luciferase. These results confirm previous in vitro studies and support the suggestion that in vivo cytosolic hsp70 and hsc70 exist mainly in an oligomer-monomer equilibrium which is dependent on the environmental temperature, the levels of ATP and the presence of denatured proteins.
European Journal of Biochemistry 02/1999; 259(1-2):505-12. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction: Microsatellites are short tandem repeat DNA sequences, comprising multiple copies of a repeat unit of 1-6 base pairs. They are common, highly polymorphic and distributed widely throughout the genome.
Recently, instability of these repeats (i.e. variations in the number of repetitive unit sequences in each microsatellite) has been reported in a variety of cancers, but not in adjacent normal tissues from the same individual (1-5).
This size instability can be demonstrated by comparing tumor DNA to normal DNA from the same individual and it is shown in the form of larger allele (expansion of the repeat element), smaller allele (deletion of the repeat element), or loss of an allele (loss of heterozygosity-LOH).
Purpose: In this study, we investigated if there is evidence of Microsatellite Instability (MI) or LOH in cancerous and adjacent normal breast tissues, with a set of 3 microsatellite markers.
The detection of either of these genetic changes in a clinical sample demonstrates the presence of a clonal population of cells that share altered genetic information, which is a characteristic of cancer cells. So, appropriately selected microssatelite loci may serve as novel markers for cancer screening (4,5).
They might also provide a sensitive indicator for generalized genomic instability and hypermutation in tumors (1).
Materials and Methods: We examined 12 matched samples from primary invasive breast carcinomas (11 of ductal type grade I,II or III and 1 of lobular type grade II) and adjacent normal tissues.
DNA was extracted from fresh-frozen tissue samples and amplified by PCR for the selected loci. We used 3 sets of primers, radiolabelled with [a- 32P]dCTP, for 3 highly informative and polymorphic microsatellite loci: two dinucleotide repeat (D17S807 and D16S301) on chromosomes 17 and 16q22.1 respesctively (12-13), and one tetranucleotide repeat (GH) on chromosome 17q22-24 (14). The markers were chosen due to their highly polymorphic nature and also because they represent different chromosome locations known to be associated with breast malignancy.
Approximately 0.2-0.3 μg of DNA was amplified in each 50-μl PCR reaction consisting of 20mM Tris-HCl(pH 8.4), 50mM KCl, 1.25mM MgCl2 , 200μM each dNTP, 1μCi [a-32P]dCTP, 0.3μg of each primer, and 5 units of Taq DNA Polymease(Gibco BRL). The thermocycle conditions were: 94oC for 30sec, 55-59oC for 1.5min and 72oC for 1.5min, for a total of 35 cycles, and a final extension step of 72oC for 10min. The PCR-products were diluted with 15μl Loading Buffer, denatured at 95oC for 5min, and analysed by electrophoresis on 6% (w/v) polyacrylamide-7M urea denaturing gels at 55W for 2.5-3h, followed by autoradiography.
Results were scored by examination of the band pattern on the autoradiograph. Microsatellite Instability is considered to have occured if either allele (band) in the tumor sample shows altered electrophoretic mobility and/or if novel alleles (bands) are present, in comparison with the normal sample. LOH in tumor specimens is characterized by loss of a band in tumor DNA compared to the normal DNA.
Results: All 12 samples studied didn’t show any instability or LOH for these particular microsatellite markers
Discussion: The frequency of instability in breast cancer varies widely in the literature, ranging from 0-85% of tumors studied. Both Lothe et al (6) and Peltomäki et al (7) showed no instances of MI in the same series of 84 tumors using the same 7 microsatellites, while Patel et al (10), at extreme odds, detected instability in 85% of tumors using 9 markers.
The greatest problem in the evaluation of the results of the different series is the fact that each group generally uses a different set of microsatellite markers, thus making true comparison impossible.
Nevertheless, our findings are in agreement with some of the reports. Wooster et al (2) found no evidence of instability (0%) in 104 breast carcinomas using one of the markers that we used (D16301). While Yee et al (9) analyzing locus D17S807, detected instability in 3 of 26 breast cancers (11.5%).
We are pursuing this study with a higher number of cases with the hope to have more conclusive results on the frequency of MI and LOH in breast cancer. It is also interesting to investigate the presence of instability in various stages of the evolution of malignant neoplasia, because the timing of MI in the multistep process of carcinogenesis may pinpoint its role either in the initiation or in the subsequent progression of cancer development. The up to now data suggest that MI may be an early event in mammary tumorigenesis (3).
Further studies on a larger series of breast specimens, using more markers, will be important in order to clarify these initial observations and to evaluate the possible diagnostic or prognostic efficiency of each microsatellite marker.
[Show abstract][Hide abstract] ABSTRACT: A recombinant virus, containing the promoter of a VL30 LTR and tagged with the neomycin gene as a selection and indicator marker, was constructed to investigate transposition events in NIH3T3 cells after SV40 transformation. This retroviral construct was transfected into psi/CRE packaging cells, and pseudovirions were used to infect NIH3T3 cells. Clones resistant to G418 bearing single-copy integrations of the recombinant virus were isolated and transformed by SV40 virus. Transpositions were detected through RFLPs with a neomycin probe and 'retrotransposition' was further confirmed by inverse PCR and DNA sequencing of transposed and parental copies. We found that: (1) retrotransposition of this recombinant virus occurred with a high frequency in a parental clone transformed with SV40 virus suggesting that the frequency of retrotransposition depended on the initial site of provirus integration; (2) the transposition frequency was independent of the transcription level of the recombinant construct; and (3) analysis of transposition-positive transformants showed that the high transposition frequency appeared to be associated with the induction of endogenous reverse transcriptases.
Biochimica et Biophysica Acta 12/1998; 1442(2-3):186-98. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microsatellites have been used for human evolution and origin studies by comparing their frequency, diversity, and allele size. In this study we report the analysis of three microsatellite loci, FMR1 CGG and flanking DXS548 and FRAXAC2, in three separate groups of the Hellenic population: Athens, representing the general Hellenic population; Epirus (northwest Greece); and Cyprus. Significant variations in frequency and diversity were found in the three groups. Compared with Athens, Epirus had a tendency for longer alleles and a higher heterozygosity for DXS548. Cyprus had a frequency of CGG alleles similar to Athens but a low heterozygosity and a limited number of alleles at DXS548 and FRAXAC2. Allele differences of microsatellite loci not only are present in remote populations but also are evident between groups belonging to the same population. Microsatellite analysis could be a useful tool for identifying the origin of the founder chromosomes in intra-population studies and the time elapsed from the establishment of each population subgroup.
Human Biology 07/1998; 70(3):621-9. · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We hereby present a rare case of a 46,XY/45,XO/47,XXY mosaic male patient with a predominance of the XY cell line. The patient, who exhibited phenotypic stigmata of both XO gonadal dysgenesis and Klinefelter syndromes, suffered from infertility and multiple urogenital abnormalities, as our investigation revealed.
Urologia Internationalis 02/1998; 61(2):111-4. · 1.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations at FRAXA and FRAXE loci are due to expansions of a CGG trinucleotide repeat and are characterized by mental retardation. Here we report a pilot screening survey by means of cytogenetic and molecular methods of 433 unrelated retarded individuals and their parents of Hellenic origin coming from various parts of Greece and Cyprus. The purpose of the study was to estimate the frequency of FRAXA mutation in individuals with nonspecific mental retardation without family history and phenotypic stigmata in the Hellenic population. Five FRAXA-positive children (1.15%) were identified, of whom four were found to carry a full mutation and one a premutation. Furthermore we present preliminary data on a screening of FRAXE mutation frequency. We screened 257 male patients with nonspecific mental retardation, finding none positive for FRAXE mutation.
[Show abstract][Hide abstract] ABSTRACT: Using transgenic mice constitutively expressing the human inducible Hsp70, we examined the role of Hsp70 on cell survival after focal cerebral ischemia. Twenty-four hours after permanent occlusion of the middle cerebral artery, no difference in infarct area was detected between Hsp70-transgenic and non-transgenic mice. In the non-transgenic mice, many pyramidal neurons of the ipsilateral hippocampus were observed to be pyknotic. However, in all Hsp70-transgenic mice, hippocampal pyramidal neurons showed normal morphology and no evidence of pyknosis. This suggests that constitutive expression of Hsp70 reduces the extent of damage following permanent middle cerebral artery occlusion.
Cell Stress and Chaperones 10/1997; 2(3):162-7. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The expansion of the trinucleotide repeat (CGG)n in successive generations through maternal meiosis is the cause of fragile X syndrome. Analysis of CA repeat polymorphisms flanking the FMR-1 gene provides evidence of a limited number of "founder" chromosomes and predisposing high-risk haplotypes related to the mutation. To investigate the origin of mutations in the fragile X syndrome in the Hellenic populations of Greece and Cyprus, we studied the alleles and haplotypes at DXS548 and FRAXAC2 loci of 16 independent fragile X and 70 normal control chromosomes. In addition, we studied 191 unrelated normal X chromosomes for the distribution and frequencies of CGG alleles. At DXS548, 6 alleles were found, 2 (194 and 196) of which were represented on fragile X chromosomes. At FRAXAC2, 6 alleles were found, 4 of which were present on fragile X chromosomes. Sixteen haplotypes were identified, but only 5 were present on fragile X chromosomes. The highest number of CGG repeats (> or = 33) were associated with haplotypes 194-147, 194-151, 194-153, and 204-155. The data provide evidence for founder chromosomes and high-risk haplotypes in the Hellenic population.
American Journal of Medical Genetics 08/1996; 64(1):234-8.
[Show abstract][Hide abstract] ABSTRACT: Heat shock treatment induces expression of several heat shock proteins and subsequent post-ischemic myocardial protection. Correlations exist between the degree of stress used to induce the heat shock proteins, the amount of the inducible heat shock protein 70 (HSP70) and the level of myocardial protection. The inducible HSP70 has also been shown to be protective in transfected myogenic cells. Here we examined the role of human inducible HSP70 in transgenic mouse hearts. Overexpression of the human HSP70 does not appear to affect normal protein synthesis or the stress response in transgenic mice compared with nontransgenic mice. After 30 min of ischemia, upon reperfusion, transgenic hearts versus nontransgenic hearts showed significantly improved recovery of contractile force (0.35 +/- 0.08 versus 0.16 +/- 0.05 g, respectively, P < 0.05), rate of contraction, and rate of relaxation. Creatine kinase, an indicator of cellular injury, was released at a high level (67.7 +/- 23.0 U/ml) upon reperfusion from nontransgenic hearts, but not transgenic hearts (1.6 +/- 0.8 U/ml). We conclude that high level constitutive expression of the human inducible HSP70 plays a direct role in the protection of the myocardium from ischemia and reperfusion injury.
Journal of Clinical Investigation 05/1995; 95(4):1854-60. · 12.81 Impact Factor