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Senescence of cardiac myocytes is frequently associated with heart diseases. To analyze senescence in cardiac myocytes, a number of biomarkers have been isolated. However, due to the complex nature of senescence, multiple markers are required for a single assay to accurately depict complex physiological changes associated with senescence. In single cells, changes in both cytoplasm and cell membrane during senescence can affect the changes in electrical impedance. Based on this phenomenon, we developed MEDoS, a novel microelectrochemical impedance spectroscopy for diagnosis of senescence, which allows us to precisely measure quantitative changes in electrical properties of aging cells. Using cardiac myocytes isolated from 3-, 6-, and 18-month-old isogenic zebrafish, we examined the efficacy of MEDoS and showed that MEDoS can identify discernible changes in electrical impedance. Taken together, our data demonstrated that electrical impedance in cells at different ages is distinct with quantitative values; these results were comparable with previously reported ones. Therefore, we propose that MEDoS be used as a new biomarker-independent methodology to obtain quantitative data on the biological senescence status of individual cells.

Background: Longstanding evidence implicates an inadequate diet as a key factor in the onset and progression of prostate cancer. The purpose herein was to discover, validate and characterize functional biomarkers of dietary supplementation capable of suppressing the course of prostate cancer in vivo., Methods: The Lady transgenic mouse model that spontaneously develops prostate cancer received a diet supplemented with a micronutrient cocktail of vitamin E, selenium and lycopene ad libitum. A proteomic analysis was conducted to screen for serum biomarkers of this dietary supplementation. Candidate peptides were validated and identified by sequencing and analyzed for their presence within the prostates of all mice by immunohistochemistry., Results: Dietary supplementation with the combined micronutrients significantly induced the expression of the megakaryocyte-specific inhibitor of angiogenesis, platelet factor-4 (P = 0.0025). This observation was made predominantly in mice lacking tumors and any manifestations associated with progressive disease beyond 37 weeks of life, at which time no survivors remained in the control group (P < 0.0001). While prostates of mice receiving standard chow were enlarged and burdened with poorly differentiated carcinoma, those of mice on the supplemented diet appeared normal. Immunohistochemical analysis revealed marked amplifications of both platelet binding and platelet factor-4 within the blood vessels of prostates from mice receiving micronutrients only., Conclusion: We present unprecedented data whereby these combined micronutrients effectively promotes tumor dormancy in early prostate cancer, following initiation mutations that may drive the angiogenesis-dependent response of the tumor, by inducing platelet factor-4 expression and concentrating it at the tumor endothelium through enhanced platelet binding.

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.

Hypoxia-inducible factor 1 (HIF-1) transactivates genes the products of which mediate tumor angiogenesis and glycolytic metabolism. Overexpression of the HIF-1 alpha subunit, resulting from intratumoral hypoxia and genetic alterations, has been demonstrated in common human cancers and is correlated with tumor angiogenesis and patient mortality. Here we demonstrate that hypoxia or HIF-1 alpha overexpression stimulates Matrigel invasion by HCT116 human colon carcinoma cells, whereas this process is inhibited by a small interfering RNA directed against HIF-1 alpha. We show that HIF-1 regulates the expression of genes encoding cathepsin D; matrix metalloproteinase 2; urokinase plasminogen activator receptor (uPAR); fibronectin 1; keratins 14, 18, and 19; vimentin; transforming growth factor alpha; and autocrine motility factor, which are proteins that play established roles in the pathophysiology of invasion. Neutralizing antibodies against uPAR block tumor cell invasion induced by hypoxia or HIF-1 alpha overexpression. These results provide a molecular basis for promotion of the invasive cancer phenotype by hypoxia and/or HIF-1 alpha overexpression.

In previous studies, we identified a common site of retroviral integration designated Fli-2 in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia cell lines. Insertion of F-MuLV at the Fli-2 locus, which was associated with the loss of the second allele, resulted in the inactivation of the erythroid cell- and megakaryocyte-specific gene p45(NFE2). Frequent disruption of p45(NFE2) due to proviral insertion suggests a role for this transcription factor in the progression of Friend virus-induced erythroleukemias. To assess this possibility, erythroleukemia was induced by F-MuLV in p45(NFE2) mutant mice. Since p45(NFE2) homozygous mice mostly die at birth, erythroleukemia was induced in +/- and +/+ mice. We demonstrate that +/- mice succumb to the disease moderately but significantly faster than +/+ mice. In addition, the spleens of +/- mice were significantly larger than those of +/+ mice. Of the 37 tumors generated from the +/- and +/+ mice, 10 gave rise to cell lines, all of which were derived from +/- mice. Establishment in culture was associated with the loss of the remaining wild-type p45(NFE2) allele in 9 of 10 of these cell lines. The loss of a functional p45(NFE2) in these cell lines was associated with a marked reduction in globin gene expression. Expression of wild-type p45(NFE2) in the nonproducer erythroleukemic cells resulted in reduced cell growth and restored the expression of globin genes. Similarly, the expression of p45(NFE2) in these cells also slows tumor growth in vivo. These results indicate that p45(NFE2) functions as an inhibitor of erythroid cell growth and that perturbation of its expression contributes to the progression of Friend erythroleukemia.

A major obstacle in the clinical management of malignant melanoma is its intrinsic resistance to chemotherapy and radiation therapy. Consequently, most patients with melanoma often do not respond to conventional anticancer therapy in a clinically significant manner. Recent advances in cancer research have provided new insights into the mechanisms of intrinsic resistance in melanomas. We have recently reported that the over-expression of tyrosinase-related protein 2 (TYRP2), an enzyme that is well characterized for its function in melanin synthesis, is associated specifically with resistance to DNA damaging drugs and radiation treatment. This review will summarize our findings as well as discuss the possible mechanisms by which TYRP2 over-expression contributes to intrinsic resistance in human malignant melanoma.

Fli-2 is a common site of proviral integration in multistage erythroleukemia cells induced by Friend murine leukemia virus (F-MuLV) or the polycythemia strain of Friend leukemia virus (FV-P). Previously, we reported that integration of Friend virus into the Fli-2 locus in CB3, an erythroleukemia cell line that harbors a homozygous inactivation of the Fli-2 locus, results in the loss of expression of two genes encoding the 45-kDa subunit of the erythroid-specific nuclear factor p45 NFE2 and the splicing factor HnRNP A1. Here, we report the identification of a third gene, Heterochromatin protein 1 (HP1alpha, also known as CBX5), which is located downstream of HnRNP A1, and p45 NFE2. Northern blot analysis revealed that the expression of HP1alpha, along with p45 NFE2 and HnRNP A1, is either undetectable or substantially reduced in CB3 cells, suggesting that HP1alpha expression is also regulated by proviral insertion within the Fli-2 locus in CB3 cells. Because p45 NFE2 was previously mapped to mouse chromosome 15, our results demonstrate that HP1alpha and HnRNP A1 are also located on mouse chromosome 15 and that the p45 NFE2, HnRNP A1, and HP1alpha genes are arranged contiguously. Contiguous arrangement of these three genes was also detected in man; this consequently localizes HP1alpha to human chromosome band 12q13.

A major obstacle in the systemic treatment of advanced malignant melanoma is its intrinsic resistance to conventionally used chemotherapeutic agents. In order to investigate the mechanisms of this intrinsic resistance, we have previously utilized retroviral insertional mutagenesis on an early-stage, drug sensitive human melanoma cell line (WM35) to establish mutated cell lines that exhibited increased resistance to cis-diammi-nedichloroplatinum(II) (CDDP). Here, we demonstrate that this increased resistance to CDDP is mediated by the over-expression of tyrosinase-related protein-2 (TYRP2), an enzyme that normally functions in the biosynthesis of the pigment, melanin. Northern and Western blot analyses revealed that the expression of TYRP2 in the virally-derived cell lines as well as in a panel of human melanoma cell lines positively correlated with their levels of resistance to CDDP. Furthermore, enforced expression of TYRP2 in WM35 cells by transfection elevated their resistance to CDDP. The increased CDDP resistance in the virally-derived clones and TYRP2 transfectants was accompanied by a reduction in CDDP-induced apoptosis. Interestingly, the virally-derived CDDP-resistant clones also showed cross resistance to carboplatin and methotrexate, but not taxol, suggesting that TYRP2 over-expression may confer resistance specifically to DNA damaging agents. Overall, these results demonstrate a novel mechanism of drug resistance in human melanoma cells that is mediated by the over-expression of TYRP2. Since TYRP2 is expressed only in cells of melanocytic lineage, this may represent the first report of a lineage-specific mechanism of drug resistance. In summary, these findings suggest a significant role for TYRP2 in the intrinsic drug resistance phenotype of human melanoma cells and may have important implications in the development of chemosensitization strategies for the clinical management of this disease.

The process of translation initiation has been postulated to play an important role in the regulation of cellular growth and proliferation. Here, we report the identification and differential expression of a fundamental translational repressor NAT1, during early postnatal cardiac development. Differential display analysis of RNA obtained from 3-day and 4-week-old rat hearts resulted in the cloning and identification of a 396 bp cDNA fragment (DRCF-6) which corresponded to the 3' terminal portion of NAT1. Northern blot analysis revealed that the mRNA expression of NAT1 was markedly elevated during the first 2 weeks of postnatal life, with an apparent peak level of expression occurring at 1 week. NAT1 mRNA levels then steadily decreased to 4 weeks of age. The NAT1 transcript has previously been shown to be extensively edited by the enzyme APOBEC-1, which deaminates specific cytidine bases to uridine; cytidine deamination at a glutamine codon (CAA) results in the formation of a stop codon (UAA) and consequently, premature termination of translation. Accordingly, Western blot analysis detected the presence of several smaller proteins in addition to the full length NAT1 protein (97 kDa), each exhibiting a distinct pattern of expression during cardiac development. APOBEC-1 editing of NAT1 during cardiac development was further supported by primer extension analysis of cytidine 1699, which was found to be predominantly edited to uridine. Immunohistochemical staining showed that NAT1 is expressed predominantly in atrial and ventricular myocytes, although staining was also detected in vascular smooth muscle cells and in the endocardium. These results suggest that NAT1 may play a role in the postnatal development of the heart and demonstrate that APOBEC-1 editing may possibly be a novel mechanism by which translation is regulated during cardiac development., (Copyright 1999 Academic Press.)

Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and other proteins involved in O2 homeostasis and tumor progression. The expression and transcriptional activity of the HIF-1alpha subunit is regulated by the cellular O2 concentration. We demonstrate that insulin, insulin-like growth factor (IGF)-1, and IGF-2 induce expression of HIF-1alpha, which is required for expression of genes encoding IGF-2, IGF-binding protein (IGFBP)-2 and IGFBP-3. These data provide a novel mechanism by which HIF-1alpha overexpression may occur in tumor cells and contribute to an autocrine growth factor loop.

Glycogenin is a self-glycosylating protein required to initiate glycogen biosynthesis. Utilizing the differential display technique to analyze changes in gene expression during early postnatal cardiac development, we have isolated and cloned a 484 bp cDNA fragment that corresponds to the 3' end of rat glycogenin. Northern blot analysis on neonatal cardiac tissues demonstrated hybridization to a 1.7-1.8 kb transcript, which was highly expressed at 3 days and at progressively reduced levels at 1, 2, 3 and 4 weeks of age. A 1624 bp fragment of rat glycogenin was cloned by RT-PCR that includes a 1002 bp open reading frame encoding a 333 amino acid protein. At the nucleotide level, rat glycogenin exhibited 87.2 and 83.6% identity with human and rabbit glycogenin over the open reading frame. The deduced amino acid sequence showed 86.7 and 83.4% identity with human and rabbit sequences, respectively. Given the significance of glycogenin in glycogen biosynthesis, the results of this study suggest a possible molecular basis for the regulation of glycogen during early postnatal cardiac development. In addition, the nucleotide and amino acid sequences of rat glycogenin may be used to investigate the physiological and pathophysiological roles of glycogenin in rat tissues.

Physiologic or pathologically induced periods of exposure to relatively low levels of oxygen during pregnancy affect the expression and function of certain genes in the placenta. In this study, the differential display technique was utilized to identify genes that are regulated in cultured cytotrophoblast cells by exposure to low levels of oxygen. Using this approach, four genes, which have been designated HRF-1, HRF-2, HRF-6, and HRF-8, were cloned and partially characterized. Northern blot analysis showed that clones HRF-1 and HRF-2 were downregulated in response to exposure to low levels of oxygen, whereas expression of HRF-6 and HRF-8 was increased. DNA sequencing and sequence analysis revealed that HRF-1 may represent an alternatively spliced or tissue-specific form of the Kruppel family zinc finger protein znfp104 gene. Clone HRF-2 showed a high degree of identity with exons 9, 10 and 11 of N33, a gene that is located within a homozygously deleted region of metastatic prostate cancer. Clones HRF-6 and HRF-8 did not exhibit significant sequence identity with known sequences in GenBank and may represent novel genes. None of these genes have previously been shown to be present in trophoblast cells, nor have their expressions been shown to be regulated by oxygen. This study demonstrates that the differential display technique is a novel and effective method to analyse oxygen-mediated changes in gene expression in trophoblast cells.

gp46 is a collagen-binding heat-shock glycoprotein with a possible role in the biosynthesis of collagen as well as in cell differentiation and fusion. In this study, the relative levels of gp46 protein and its mRNA transcript were examined, as well as the mRNA levels of collagen types I and IV in first trimester and term human placental tissues. Western blot analysis revealed substantially higher levels of gp46 in first trimester placentae than in term placentae. Similarly, elevated levels of type IV collagen transcript were detected in first trimester relative to term issues. Interestingly, the levels of gp46 and type I collagen mRNA remained unchanged. Immunohistochemical analysis of first trimester tissues demonstrated intense gp46 staining in mononucleated villous and extravillous cytotrophoblasts, decidual cells and in the villous connective tissue stroma. Syncytiotrophoblast in the same tissues also exhibited gp46 staining but at a reduced intensity. In chronic villi of term placentae, faint gp46 staining was only observed in the syncytiotrophoblast layer. However, as in the first trimester placentae, intense labelling was evident in the extravillous cytotrophoblasts and decidual cells of these tissues. These results suggest a developmental regulation of gp46 expression at the fetal-maternal interface during pregnancy and suggest a possible functional link between gp46 and collagen type IV. during gestation.

Collagen-binding heat-shock proteins of M(r) 46-47 kDa have been postulated to function as putative molecular chaperones in the biosynthesis of collagen in several species. The rat homologue of this family of heat-shock proteins is called gp46. In the present study, we employed Western blotting and immunohistochemical methods to determine the tissue distribution and cellular localization of gp46 in the thoracic aorta, heart, kidney, liver and lung of eight-day-old Wistar rats. Highest levels of gp46 were detected in the thoracic aorta and lung, followed by the kidney and heart. Gp46 levels were low to undetectable by Western blot analysis in the liver. Immunohistochemistry revealed that gp46 labelling was observed almost exclusively in three distinct cell types: fibroblasts, muscle cells, and some epithelial cells. Gp46 was detected in the fibroblasts of the hepatic triad, in the interstitium of the alveolar wall and in the tunica adventitia of blood vessels in the majority of tissues examined, in atrial and ventricular cardiomyocytes, in vascular smooth muscle cells of the abluminal portion of the tunica media, in parietal epithelial cells and mesangial cells of the glomerulus, in epithelial cells of the distal tubules and collecting ducts in the kidney and clusters of immature renal tubules, in epithelial cells of the bile duct, and in mesodermal cells surrounding the liver. These results demonstrate that gp46 is present in collagen producing cells and cells undergoing rapid growth and development, suggesting that gp46 may play a significant role in these processes.

The temporal expression of protein and mRNA encoding the collagen-binding heat-shock glycoprotein, gp46, were determined in the heart, kidney, and lung during early rat postnatal development. The steady-state levels of collagen types I and IV mRNA expression were also examined to determine if gp46 and these collagen types are co-regulated during ontogenesis. Western blot analysis using a monoclonal antibody to gp46 revealed that gp46 levels are developmentally regulated. In heart and kidney, gp46 levels were high on days 3 and 8, reduced significantly on day 25, and low to undetectable on day 69. Protein levels of gp46 in the lung exhibited a similar temporal pattern except on day 3, when very low levels of gp46 were detected. mRNA expression of gp46 during early postnatal development did not correlate with gp46 protein accumulation in these tissues, suggesting a complex pre- and post-translational regulatory scheme. In the heart, protein levels of gp46 correlated well with collagen type I alpha 1(I) mRNA expression but not with collagen type IV alpha 1(IV). In contrast, gp46 protein levels closely paralleled alpha 1(IV) expression in the kidney. Gp46 levels exhibited no apparent correlation with either alpha 1(I) or alpha 1(IV) levels in the lung. These results show that gp46 is developmentally regulated at both the protein and mRNA levels in a tissue specific manner. The relationship between gp46 and collagen alpha 1(I) and alpha 1(IV) chain mRNA expression also has been shown to be tissue specific.