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Helicobacter pylori infects 50% of mankind. The vast majority of H. pylori infection occurs in the developing countries where up to 80% of the middle-aged adults may be infected. Bacterial infection causes an inflammatory response that proceeds through a series of intermediated stages of precancerous lesions (gastritis, atrophy, intestinal metaplasia, and dysplasia). Among infected individuals, approximately 10% develops severe gastric lesions such as peptic ulcer disease, 1-3% progresses to gastric cancer (GC) with a low 5-year survival rate, and 0.1% develops mucosa-associated lymphoid tissue (MALT). GC is one of the most common cancer and the third leading cause of cancer-related deaths worldwide. In this review, we have summarized the most recent papers about molecular mechanisms of H. pylori pathogenesis. The main important steps of H. pylori infection such as adhesion, entry in epithelial gastric cells, activation of intracellular pathways until epigenetic modifications have been described.

The aim of this study was to demonstrate in the adrenocortical and renal tissues of two species of frog, Rana italica and Rana esculenta, the presence and distribution of five neuropeptides: atrial natriuretic peptide (ANP), Leu-enkephalin (Leu-ENK), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal peptide (VIP). In anurans, the adrenal medulla is the site for the synthesis, storage and secretion of not only catecholamines but also various peptides. These peptides should not be regarded only as neurotransmitters or modulators for the secretion of catecholamines, but also as hormonal substances that induce systemic effects. All the peptides studied (ANP, Leu-ENK, NPY, SP and VIP) are present in both organs. However, different patterns of expression were observed for some of the peptides in two frogs. Immunopositivity to ANP was found in small clusters of chromaffin cells in both frogs whereas a clear strong positivity was present only in Rana esculenta kidney. Large clusters of chromaffin cells were immunoreactive to Leu-ENK in Rana italica but there were approximately 25% fewer compared to the positive cells present in Rana esculenta. Epithelial cells of renal tubules showed strong immunopositivity to Leu-ENK in Rana esculenta but not in Rana italica. A large number of adrenal cells (70-80%) were immunoreactive to NPY in Rana italica, while in Rana esculenta this peptide was localized in small clusters of chromaffin cells. Both frogs showed many NPY-positive cells in kidney. Many chromaffin cells were found positive to SP and VIP. A strong positivity was also observed in kidney in both frogs. These observations suggest a possible role of these peptides in the control of the physiological functions of adrenal glands and kidney of the two species of frogs studied.

The adrenal gland of the lizard Podarcis sicula is formed by a dorsal ribbon of chromaffin cells, generally defined as medullary tissue, arranged along a central part of steroidogenic cells considered as cortical tissue. These two tissues produce catecholamines and steroids as part of the hypothalamo-hypophyseal-adrenal gland axis. Recent studies have demonstrated that Podarcis sicula adrenal gland is not only under hypothalamo-hypophyseal axis control but that several peptides may influence the physiological activity of the gland; among these, vasoactive intestinal peptide is able to enhance strongly both catecholamine and steroid hormone production. The aim of the present study was to verify whether vasoactive intestinal peptide administration could become deleterious. For this reason, we monitored the pattern of expression of two members of the Bcl-2 family, Bcl-2 and Bax, in control and vasoactive intestinal peptide treated specimens. Furthermore, we also tested if peptide treatment induces apoptosis by TUNEL assay.

To determine whether the molecular components implicated in the regulation of the cell cycle are activated in myocytes after infarction, the expression of cyclins E, A, and B and the levels of their associated kinase activity were measured at 1 and 7 days following surgery. The quantity of cdk2 and cdc2 and the level of their kinase activity were also determined. Myocardial infarction was characterized by an increase in cyclins E, A, and B and cdc2 proteins in the surviving myocytes at 1 and 7 days. Cyclin E, A, and B and cdk2 and cdc2 kinase activity also increased. The quantity of cyclins E and A and the level of cyclin E-associated kinase activity in myocytes after infarction were comparable with those measured in neonatal myocytes. Moreover, cdc2 protein and cdc2 kinase activity in myocytes reached levels after infarction which were similar to those in neonatal myocytes. Thus, myocytes react to myocardial infarction by activating cyclins and cyclin-dependent kinases which may be coupled with the regeneration of muscle mass and recovery of ventricular function.

Alkylphenols (AP) are widespread environmental compounds belonging to the large family of substances known as Endocrine Disrupting Chemicals (EDCs). The present study was carried out to assess the effects of Octylphenol (OP) alone and in combination with Nonylphenol (NP) on the hypothalamus-pituitary-adrenal gland (HPA) axis of the lizard Podarcis sicula. Lizards are good bioindicators due to their features such as wide distribution, large population and good sensitivity to contaminants. Results obtained showed a time and dose-dependent stimulation of the HPA together with a high variation of both catecholamine plasma levels and greater vascularization and hypertrophy of steroidogenic cord of adrenal gland after both OP and OP + NP treatments. Interestingly, the OP + NP mixture treatment has provoked a state of stress of the adrenal gland which in fact appeared to be characterized by the presence of a marked macrophage infiltration which can be seen especially close to the connective capsule surrounding the gland. This macrophage infiltration could be an evidence of a particularly pronounced inflammatory state to indicate, probably, an animal’s response to a non-physiological situation.

The introduction of HAART (highly-active-antiretroviral-therapy) has resulted in extended survival of HIV positive patients. Conversely, due to the prolonged expectancy of life and the ageing of the HIV positive population, tumors are now one of the major cause of death, and among them hepatocellular carcinoma (HCC) has become a growing concern in these patients. Considering the potential anti-tumoral effects of HIV protease inhibitors, we decided to evaluate the anti-tumoral activity of Amprenavir on liver carcinoma and to evaluate its potential synergistic effects in combination with standard chemoterapic drugs, such as Doxorubicin. Our results indicate that Amprenavir had direct inhibitory effects on invasion of Huh-7 hepatocarcinoma cell lines, inhibiting MMP proteolytic activation. Amprenavir was able to delay the growth of hepatocarcinoma xenografts in nude mice and had a synergistic effect with Doxorubicin. Furthermore, Amprenavir was able to promote regression of hepatocarcinoma growth in vivo by anti-angiogenetic and overall anti-tumor activities, independently by PI3K/AKT related pathways that at today is one of the more suggestive hypothesis to explain the anti-tumor effects of the different protease inhibitors. In summary these findings suggest novel anti-neoplastic action of Amprenavir on liver cancer showing the possibility of novel combination therapies. J. Cell. Physiol. 228: 640–645, 2013. © 2012 Wiley Periodicals, Inc.

In the last few years, a major goal of cardiac research has been to drive stem cell differentiation to replace damaged myocardium. Several research groups have attempted to differentiate potential cardiac stem cells (CSCs) using bi- or three-dimensional systems supplemented with growth factors or molecules acting as differentiating substances. We hypothesize that these systems failed to induce a complete differentiation because they lacked an architectural space. In the present study, we isolated a pool of small proliferating and fibroblast-like cells from adult rat myocardium. The phenotype of these cells was assessed and the characterized cells were cultured in a collagen I/OPLA scaffold with horse serum to obtain fine myocardial differentiation. C-Kit(POS)/Sca-1(POS) CSCs fully differentiated in vitro when an environment more similar to the CSC niche was created. These experiments demonstrated an important model for the study of the biology of CSCs and the biochemical pathways that lead to myocardial differentiation. The results pave the way for a new surgical approach.

The aim of this study was to investigate the distribution and function of VIP in the adrenal gland of the lizard, Podarcis sicula. We have shown by immunohistochemistry that VIP fibers were localized exclusively around clusters of chromaffin cells in the dorsal ribbon of the lizard adrenal gland. Moreover, a strong positivity for this peptide was observed within ganglial cells and within most chromaffin cells of the gland. To investigate the effects of VIP on the adrenal gland, we have treated lizards with several doses of this peptide and we have shown that injections of exogenous VIP increased plasma levels of catecholamines and corticosteroids, but not of ACTH. This probably suggests a direct effect of VIP on the control of adrenal hormone secretion without the involvement of the hypothalamo-hypophyseal axis. Our results also establish that the increased levels of the hormones were modulated in a time- and dose-dependent manner. Therefore, our morphological studies showed a clear increased function of steroidogenic cells. In the medullary region, VIP administration induced not only a functional enhancement of adrenaline release from adrenergic cells, but also a shift of noradrenaline cells to adrenaline ones.

Differential gene expression of cell lines derived from a malignant melanoma or its autologous lymph node metastasis using cDNA arrays indicated down-regulation of PRSS11, a gene encoding the serine protease HtrA1, a homolog of the Escherichia coli protease HtrA, in the metastatic line. Stable PRSS11 overexpression in the metastatic cell line strongly inhibited proliferation, chemoinvasion and Nm23-H1 protein expression in vitro, as well as cell growth in vivo in nu/nu mice. A polyclonal anti- HtrA1 serum demonstrated a significantly higher expression in primary melanomas when compared to unrelated metastatic lesions in a human melanoma tissue array, and down-modulation of HtrA1 expression in autologous lymph node melanoma metastases in seven out of 11 cases examined. These results suggest that down-regulation of PRSS11 and HtrA1 expression may represent an indicator of melanoma progression.

Myogenic transcription is repressed in myoblasts by serum-activated cyclin-dependent kinases, such as cdk2 and cdk4. Serum withdrawal promotes muscle-specific gene expression at least in part by down-regulating the activity of these cdks. Unlike the other cdks, cdk9 is not serum- or cell cycle-regulated and is instead involved in the regulation of transcriptional elongation by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. While ectopic expression of cdk2 together with its regulatory subunits (cyclins E and A) inhibits myogenic transcription, overproduction of cdk9 and its associated cyclin (cyclin T2a) strengthens MyoD-dependent transcription and stimulates myogenic differentiation in both MyoD-converted fibroblasts and C2C12 muscle cells. Conversely, inhibition of cdk9 activity by a dominant negative form (cdk9-dn) represses the myogenic program. Cdk9, cyclinT2 and MyoD can be detected in a multimeric complex in C2C12 cells, with the minimal cdk9-binding region of MyoD mapping within 101-161 aa of the bHLH region. Finally, cdk9 can phosphorylate MyoD in vitro, suggesting the possibility that cdk9/cycT2a regulation of muscle differentiation includes the direct enzymatic activity of the kinase on MyoD.

TheWFS1gene, encoding a transmembrane glycoprotein of the endoplasmic reticulum called wolframin, is mutated in Wolfram syndrome, an autosomal recessive disorder defined by the association of diabetes mellitus, optic atrophy, and further organ abnormalities. Disruption of theWFS1gene in mice causes progressiveβ-cell loss in the pancreas and impaired stimulus-secretion coupling in insulin secretion. However, little is known about the physiological functions of this protein. We investigated the immunohistochemical expression of wolframin in human placenta throughout pregnancy in normal women and diabetic pregnant women. In normal placenta, there was a modulation of wolframin throughout pregnancy with a strong level of expression during the first trimester and a moderate level in the third trimester of gestation. In diabetic women, wolframin expression was strongly reduced in the third trimester of gestation. The pattern of expression of wolframin in normal placenta suggests that this protein may be required to sustain normal rates of cytotrophoblast cell proliferation during the first trimester of gestation. The decrease in wolframin expression in diabetic placenta suggests that this protein may participate in maintaining the physiologic glucose homeostasis in this organ.

Cdk9, previously known as PITALRE, belongs to the Cdc2 family of protein kinases. We report the isolation and characterization of the complete gene coding for the murine Cdk9 protein. The gene contains seven exons spanning over 6 kb of genomic DNA, and the exon/intron boundaries conformed to the GT/AG rule. The Cdk9 gene mapped on mouse chromosome 2, which is consistent with the known region of synteny with human chromosome 9q34.1. The length of the individual exons ranged from 82 to 850 bp, and introns ranged from 452 to 1,465 bp. The further 5' flanking region of the gene showed features of a housekeeping promoter, such as the lack of a canonical TATA box and the presence of a CCAAT box as well as several GC boxes, which are potential binding sites for numerous transcription factors. Additionally, we performed a basic analysis of the transcriptional activity of the promoter and found that the 364 bp of Cdk9 5' flanking region were able to elicit high transcriptional levels of a luciferase reporter gene in NIH3T3 cells. This study provides the molecular basis for understanding the transcriptional control of the Cdk9 gene, and could serve to facilitate the molecular genetic investigation of Cdk9 function during mouse embryonal development.

Employing the myeloblastic leukemia M1 cell line, which does not express endogenous p53, and genetically engineered variants, it was recently shown that activation of p53, using a p53 temperature-sensitive mutant transgene (p53ts), resulted in rapid apoptosis that was delayed by high level ectopic expression of bcl-2. In this report, advantage has been taken of these M1 variants to investigate the relationship between p53-mediated G1 arrest and apoptosis. Flow cytometric cell cycle analysis has provided evidence that activation of wild-type (wt) p53 function in M1 cells resulted in the induction of G1 growth arrest; this was clearly seen in the M1p53/bcl-2 cells because of the delay in apoptosis that unmasked p53-induced G1 growth arrest. This finding was further corroborated at the molecular level by analysis of the expression and function of key cell cycle regulatory genes in M1p53 versus M1p53/bcl-2 cells after the activation of wt p53 function; events that take place at early times during the p53-induced G1 arrest occur in both the M1p53 and the M1p53/bcl-2 cells, whereas later events occur only in the M1p53/bcl-2 cells, which undergo delayed apoptosis, thereby allowing the cells to complete G1 arrest. Finally, it was observed that a spectrum of p53 target genes implicated in p53-induced growth suppression and apoptosis were similarly regulated, either induced (gadd45, waf1, mdm2, and bax) or suppressed (c-myc and bcl-2), after activation of wt p53 function in M1p53 and M1p53/bcl-2 cells. Taken together, these findings show that wt p53 can simultaneously induce the genetic programs of both G1 growth arrest and apoptosis within the same cell type, in which the genetic program of cell death can proceed in either G1-arrested (M1p53/bcl-2) or cycling (M1p53) cells. These findings increase our understanding of the functions of p53 as a tumor suppressor and how alterations in these functions could contribute to malignancy.

The introduction of highly active antiretroviral therapies (HAART) has significantly changed the clinical course of HIV disease, with prolonged survival and better quality of life for HIV infected patients. However, this successful therapeutic advance has been partially marked by the development of serious long-term side effects including metabolic alterations, cardiovascular disease, kidney impairment, bone alterations and adipose tissue redistribution. This last phenomenon is currently indicated as HIV related lipodystrophy (Barbaro, 2006). Even if some studies suggested an independent role for HIV in the development of lipodystrophic phenotype, there is a widely accepted consensus that the risk to develop fat redistribution in HIV patients has to be mostly related to antiretroviral therapy. In order to investigate new pathways involved in the development of lipodystrophy, our group performed an array screening using two identical filter arrays with cDNAlabeled probes, generated from the adipose tissue of either HIV patients affected or not affected by lipodystrophy. Among the genes selected, we focused our attention on a recently described 48 kDa protein of 417 amino acids named FAP48. Our results suggest, using 3T3-L1-FAP48 stable clone, that FAP48 over-expression results in rapid NFAT dephosphorilatyon by activating CaN and in the increase of aP2 gene transcription, a gene expressed at the last phase of the adipocyte differentiation. These data support the role of Fap48 in the activation of adipocyte differentiation through a pathway involving NFAT. Moreover we evaluated the expression of PPARγ and aP2 in 3T3-L1 FAP48pcDNA stably transfected cells treated with five antiretroviral drugs (Indinavir, Amprenavir, Efavirenz, Stavudine and Saquinavir), belonging to the three main classes of anti-HIV drugs, that were able, in our experimental model, to affect adipocyte differentiation (Esposito et al., 2009). We observed that cells treated with Saquinavir and Efavirenz, using 3T3-L1FAP48 stable clone, are characterized by an increased expression of PPARγ and aP2, during the 6 day time course, compared with the control cells. This evidence supports the hypothesis of a protective mechanism, that in 3T3L1 cells could counteract the toxicity of Efavirenz and Saquinavir or could be activated in presence of these drugs. Drawing from our experimental results it can be then postulated that this mechanism could work trough FAP48/ FBP52/Hsp90 pathway, suggesting this complex as a potential target for novel therapeutic approaches to the HAART related lipodystrophy in patients treated with regimen including Efavirenz and Saquinavir. references

The cell cycle consists of a number of complex biochemical pathways that ensure that the start of a particular event depends on the successful and right end of previous steps in the pathway. An important role is played by cyclin/cyclin-dependent kinase (cdk) complexes which are critical regulators of cell cycle progression and RNA transcription. To ensure proper progression through each phase, cells have developed a series of orchestrated checkpoints that govern the different cellular kinases required for distinct cell cycle events. In particular, several cell cycle protein kinases, including members of the Aurora family and the Polo-like kinases, play critical roles in mitotic entry and chromosome segregation that ensure the correct formation of daughter cells. Tumour cell proliferation is frequently associated to both genetic and epigenetic mechanisms commonly affecting the expression of cell cycle regulatory proteins or causing an incompetent checkpoint control, resulting in aberrant responses to cellular damage. These alterations result not only in proliferative advantages but also in an increased susceptibility to the accumulation of additional genetic alterations that contribute to tumour progression and acquisition of more aggressive phenotypes. In the last years, the identification of anticancer drugs directed against critical cell cycle regulators has received particular attention. Specifically, several preclinical and clinical trials are addressing cdks or cell cycle protein kinase inhibitors. Starting from a description of cell cycle, this review summarizes the most recent studies on drugs targeting cell cycle regulators that are being used in cancer therapy.

It is known that the MyoD family members (MyoD, Myf5, myogenin, and MRF4) play a pivotal role in the complex mechanism of skeletal muscle cell differentiation. However, fragmentary information on transcription factor-specific regulation is available and data on their post-transcriptional and post-translational behavior are still missing. In this work, we combined mRNA and protein expression analysis with their subcellular localization. Each myogenic regulator factor (MRF) revealed a specific mRNA trend and a protein quantitative analysis not overlapping, suggesting the presence of post-transcriptional mechanisms. In addition, each MRF showed a specific behavior in situ, characterized by a differentiation stage-dependent localization suggestive of a post-translational regulation also. Consistently with their transcriptional activity, immunogold electron microscopy data revealed MRFs distribution in interchromatin domains. Our results showed a MyoD and Myf5 contrasting expression profile in proliferating myoblasts, as well as myogenin and MRF4 opposite distribution in the terminally differentiated myotubes. Interestingly, MRFs expression and subcellular localization analysis during C2C12 cell differentiation stages showed two main MRFs regulation mechanisms: (i) the protein half-life regulation to modulate the differentiation stage-dependent transcriptional activity and (ii) the cytoplasmic retention, as a translocation process, to inhibit the transcriptional activity. Therefore, our results exhibit that MRFs nucleo-cytoplasmic trafficking is involved in muscle differentiation and suggest that, besides the MRFs expression level, also MRFs subcellular localization, related to their functional activity, plays a key role as a regulatory step in transcriptional control mechanisms.

Histone deacetylase (HDAC) inhibitors are promising new epi-drugs, but the presence of both class I and class II enzymes in HDAC complexes precludes a detailed elucidation of the individual HDAC functions. By using the class II-specific HDAC inhibitor MC1568, we separated class I- and class II-dependent effects and defined the roles of class II enzymes in muscle differentiation in cultured cells and in vivo. MC1568 arrests myogenesis by (i) decreasing myocyte enhancer factor 2D (MEF2D) expression, (ii) by stabilizing the HDAC4-HDAC3-MEF2D complex, and (iii) paradoxically, by inhibiting differentiation-induced MEF2D acetylation. In vivo MC1568 shows an apparent tissue-selective HDAC inhibition. In skeletal muscle and heart, MC1568 inhibits the activity of HDAC4 and HDAC5 without affecting HDAC3 activity, thereby leaving MEF2-HDAC complexes in a repressed state. Our results suggest that HDAC class II-selective inhibitors might have a therapeutic potential for the treatment of muscle and heart diseases. Histone deacetylase (HDAC) inhibitors are promising new epi-drugs, but the presence of both class I and class II enzymes in HDAC complexes precludes a detailed elucidation of the individual HDAC functions. By using the class II-specific HDAC inhibitor MC1568, we separated class I- and class II-dependent effects and defined the roles of class II enzymes in muscle differentiation in cultured cells and in vivo. MC1568 arrests myogenesis by (i) decreasing myocyte enhancer factor 2D (MEF2D) expression, (ii) by stabilizing the HDAC4-HDAC3-MEF2D complex, and (iii) paradoxically, by inhibiting differentiation-induced MEF2D acetylation. In vivo MC1568 shows an apparent tissue-selective HDAC inhibition. In skeletal muscle and heart, MC1568 inhibits the activity of HDAC4 and HDAC5 without affecting HDAC3 activity, thereby leaving MEF2-HDAC complexes in a repressed state. Our results suggest that HDAC class II-selective inhibitors might have a therapeutic potential for the treatment of muscle and heart diseases.

Recently, suggestive evidence for piecemeal degranulation (PMD), a particulate pattern of cell secretion accomplished by vesicle-mediated extracellular transport of granule-stored material, has been provided by electron microscopy investigations in chromaffin cells of different vertebrate species. In this study, chromaffin tissue from the interrenal gland of the amphibian urodede Triturus carnifex has been investigated by quantitative transmission electron microscopy in search for ultrastructural clues indicative of a vesicle-mediated mode of cell degranulation. Interestingly, a single type of chromaffin cell is recognizable in T. carnifex, which undergoes seasonal variations in its adrenaline and noradrenaline granule ratio according to an annual cycle that matches the trend of changes in secondary sexual characters. In this study, we looked for a series of ultrastructural changes regarded as highly specific for PMD. We calculated the percentage of (i) resting, unaltered granules (type 1 granules); (ii) granules with changes indicative of progressive release of secretory materials, that is, granules with lucent areas in their cores, reduced electron density, disassembled matrices, and residual cores (type 2 granules); and (iii) membrane empty containers (type 3 granules) in chromaffin cells of T. carnifex during the annual cycle. We found a significant increase in type 2 and 3 granules, accompanied by a significant decrease in type 1 granules, in the April and November samples. During the same seasonal periods, the number per net cytoplasmic area of 30-100-nm diameter electron-dense vesicles was found to be significantly augmented, and there was also an augmented percentage of chromaffin granules showing blebs or protrusions in their profiles. These ultrastructural data are indicative of an increased vesicle-mediated transport of chromaffin granule products for extracellular release in the amphibian T. carnifex in accordance with the increased rate of catecholamine release. This vesicle-mediated pattern of cell secretion suits the schema of PMD. In an evolutionary perspective, these findings suggest that PMD is a secretory pathway that has been highly conserved throughout vertebrate classes. Anat Rec, 2009. (c) 2008 Wiley-Liss, Inc.

none 11 Heat shock protein B (HspB) is one of the dominant proteins recognized by most Helicobacter pylori-infected persons and is being considered as potential candidates for subunit vaccines. In the present study we describe the generation of an antibody against HspB and its use in immunohistochemical assays on gastric biopsies. We have demonstrated that our rabbit polyclonal antibody against HspB did not recognize any protein in lysates from a lung human epithelial cell (H1299) line and did not cross-react with the other members of human heat shock proteins. Secondly, we have observed that in gastric biopsies, HspB immunostaining was present inside the cytoplasm of human epithelial cells with a particular localization in the apical portion of gastric epithelial cells other than in the extracellular spaces among gastric cells of human stomach. Finally, we have demonstrated a cytoplasmic HspB immunostaining in groups of neoplastic cells of MALT lymphoma. In conclusion, our observations suggest a possible involvement of HspB in the pathogenesis of H. pylori-related pathologies such as gastritis, ulcer and gastric cancer. © 2008 Wiley-Liss, Inc. mixed DE LUCA A; DE FALCO M; MANENTE L; DATTILO D; LUCARIELLO A; ESPOSITO V; GNARINI M; CITRO G; BALDI A; TUFANO MA; IAQUINTO G De Luca A.; De Falco M.; Manente L.; Dattilo D.; Lucariello A.; Esposito V.; Gnarini M.; Citro G.; Baldi A.; Tufano M.A.; Iaquinto G.

a Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, United States b Department of Biological Science, Section of Evolutive and Comparative Biology, University of Naples ‘‘Federico II’’, Italy c Department of Cardiac Physiopathology, University of Buenos Aires, Argentina d Department of Medicine and Public Health, Sect. of Clinical Anatomy, Second University of Naples, Italy e Department of Biochemistry, Sect. of Pathologic Anatomy, Second University of Naples, Italy

One strategy in the development of anticancer therapeutics has been to arrest malignant proliferation through inhibition of the enzymatic activity of cyclin-dependent kinases (cdks), which are key regulatory molecules of the cell cycle. Over the past few years, numerous compounds with remarkable cdk inhibitory activity have been studied in cancer therapy, although it is very difficult to point out the best cdk to target. An excellent candidate appears to be cdk2, whose alteration is a pathogenic hallmark of tumorigenesis. The small molecule described in our study showed an inhibitory effect on the kinase activity of cdk2, a significant growth arrest observed in a colony formation assay and a reduction in the size of the tumor in nude mice, thus suggesting its potential role as a promising new type of mechanism-based antitumor drug, also for the treatment of hyperproliferative disorders.

With the aim to find novel partners of human Cyclin T2a, we performed a two-hybrid screening in yeast using the full-length cDNA of this cyclin as bait, and a human heart cDNA library as preys Source. Upon several interesting genes selected, our attention has been focused on the cDNA coding for PKN alpha, a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. Co-immunoprecipitation and in vitro pull-down assays independently confirmed the interaction between the two proteins. Luciferase assays, performed on NIH3T3 cell extracts after transfection with a MyoD-responsive promoter, pointed out that PKN alpha was able to enhance MyoD-dependent transcription, and that this effect was further increased when cyclin T2a was co-overexpressed. Finally, overexpression of both Cyclin T2a and PKNC alpha. in C2C12 cells strongly enhanced the expression of myogenic differentiation markers, such as Myogenin and Myosin Heavy Chain, during starvation-induced differentiation. Taken together, our data strengthen the hypothesis that cyclin T2a plays a role in muscle differentiation, and propose PKNa as a novel partner of Cyclin T2a in this process.

Purpose: Accumulating evidences show a higher incidence of hepatic neoplasm in HIV/hepatitis C virus (HCV)–coinfected individuals compared with HCV-monoinfected patients. Treatment with HIV-1 protease inhibitors inhibited cancer-promoted angiogenesis in HIV-infected patients affected by Kaposi sarcoma. We aimed to evaluate the antineoplastic potential activities of the protease inhibitor indinavir (Crixivan) in in vitro and in vivo hepatocarcinoma models. Experimental Design: We analyzed effects of indinavir on cell growth and invasiveness in Huh7 and SK-HEP-1 hepatocarcinoma cell lines and on in vivo tumor growth of the same cells in nude mice. Morphologic and molecular analyses on explanted tumors were carried out to evaluate vascularization and apoptosis. Results: We observed a reduced ability to invade an in vitro extracellular matrix for both cell lines treated with indinavir compared with controls (P = 0,001). Moreover, indinavir treatment was able to inhibit matrix metalloproteinase-2 proteolytic activation, whereas there was no effect on cell proliferation. The drug was also able to delay in vivo tumor growth. The inhibition of tumor growth was statistically significant from days 6 to 21 (P = 0.004 and P = 0.003, respectively). Moreover, the drug showed antiangiogenic and proapoptotic actions, as revealed by vessel count and apoptotic index by terminal deoxynucleotide transferase–mediated nick end labeling in explanted tumors. Finally, treatment with indinavir did not block the production of vascular endothelial growth factor in the tumors. Conclusion: Indinavir could be helpful to prevent the development of hepatocarcinomas in HIV/HCV–coinfected individuals. In view of the current trend to substitute protease inhibitors with other antiretroviral agents, this information may have clinical implications.

Myocyte differentiation is due to transcription of genes that characterize the phenotypic and biochemical identity of differentiated muscle cells. These are the myogenic regulatory factors (MRFs) MyoD, Myf5, myogenin and MRF4. Overexpression of cdk/cyclins has been reported to inhibit the activity of MyoD and prevent myogenic differentiation by different modalities. Unlike other cdk/cyclin complexes, overexpression of cdk9/cyclin T2a, enhances MyoD function and promotes myogenic differentiation. In addition, cyclin T2a interacting with a novel partner, PKN alpha, is able to strongly enhance the expression of myogenic differentiation markers, such as myogenin and Myosin Heavy Chain. So, cyclin T2a could stimulate myogenic differentiation interacting with different kinase partners Cdk9 or PKN alpha in a synergistic or antagonistic way.

Purpose: Employing an in vitro model system of human melanoma progression, we previously reported ferritin light chain (L-ferritin) gene overexpression in the metastatic phenotype. Here, we attempted to characterize the role of ferritin in the biology of human melanoma and in the progression of this disease. Experimental Design: Starting from the LM human metastatic melanoma cell line, we engineered cell clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. These cells were then assayed for their growth capabilities, chemoinvasive properties, and sensitivity to oxidative stress. Additionally, ferritin protein content in primary and metastatic human melanomas was determined by immunohistochemistry. Results: Artificial L-ferritin down-regulation in the LM cells strongly inhibited proliferation and chemoinvasion in vitro and cell growth in vivo. In addition, L-ferritin down-regulated cells displayed enhanced sensitivity to oxidative stress and to apoptosis. Concurrently, immunohistochemical analysis of a human melanoma tissue array revealed that ferritin expression level in metastatic lesions was significantly higher (P < 0.0001) than in primary melanomas. Furthermore, ferritin expression was constantly up-regulated in autologous lymph node melanoma metastases when compared with the respective primary tumors in a cohort of 11 patients. Conclusions: These data suggest that high ferritin expression can enhance cell growth and improve resistance to oxidative stress in metastatic melanoma cells by interfering with their cellular antioxidant system. The potential significance of these findings deserves to be validated in a clinical setting.

Apelin is a novel bioactive peptide that has been isolated from bovine stomach extracts and identified as the endogenous ligand for the APJ receptor. Although the main physiological functions of apelin have not yet been clarified, it is known that apelin is involved in the regulation of blood pressure, central control of body fluid homeostasis and the modulation of immune response. In order to investigate the distribution of apelin in reptiles, we have performed an immunohistochemical analysis on tissues of the lizard Podarcis sicula. The peptide was found to be widely distributed, although its cellular localization differed in the various organs examined. A strong immunopositivity was found in the heart, stomach and intestine. In the spleen, an intense apelin immunopositivity was restricted to a discrete number of cells scattered throughout the red pulp and co-localized with immunoglobulin kappa and lambda chains, suggesting an analogous function of this peptide in immune responses also in reptiles. Intriguingly, apelin immunoreactivity was discretely localized in endothelial cells in the lung and thyroid gland. In the light of these data, we conclude that apelin may have multiple functions in reptiles.

The human HtrA family of proteases consists of four members: HtrA1, HtrA2, HtrA3, and HtrA4. In humans the four HtrA homologues appear to be involved in several important functions such as cell growth, apoptosis, and inflammatory reactions, and they control cell fate via regulated protein metabolism. In previous studies it was shown that the expression of HtrA1 was ubiquitous in normal adult human tissues. Here we examined the expression of HtrA1 protein and its corresponding mRNA during mouse embryogenesis using Northern blotting hybridization, RT-PCR, and immunohistochemical staining analyses. Our results indicate that HtrA1 is expressed in a variety of tissues in mouse embryos. Furthermore, this expression is regulated in a spatial and temporal manner. Relatively low levels of HtrA1 mRNA are detected in embryos at the beginning of organogenesis (E8), and the levels of expression increase during late organogenesis (E14-E19). Our results show that HtrA1 was expressed during embryonic development in specific areas where signaling by TGFβ family proteins plays important regulatory roles. The expression of HtrA1, documented both at mRNA and protein levels by RT-PCR and immunohistochemistry in the developing nervous system, is consistent with a possible role of this protein both in dividing and postmitotic neurons, possibly via its documented inhibitory effects on TGFβ proteins. An exhaustive knowledge of the different cell- and tissue-specific patterns of expression of HtrA1 in normal mouse embryos is essential for a critical evaluation of the exact role played by this protein during development.

The adenoviral E1A proteins have been implicated in promotion of proliferation and transformation, inhibition of differentiation, induction of apoptosis, regulation of transcription, and suppression of tumor growth. The ability of E1A to override the fundamental controls of host cells is based on its ability to physically interact with several cellular proteins. We recently characterized RACK1 as a new E1A-interacting protein. In this report, we show that the extreme N-terminal region of E1A, spanning from aminoacids 1-36, and the conserved WD regions of RACK1 are responsible for this interaction. We also demonstrate that E1A and RACK1 colocalize at the perinuclear membrane in the cells. Furthermore, we provide evidence that E1A is able to antagonize the inhibitory effects of RACK1 on Src activity. These results suggest that RACK1 signaling pathway may be a functional target of E1A, contributing to E1A oncogenic effect in the host cells. © 2003 Wiley-Liss, Inc.

The balance between acetylation and deacetylation of histone and nonhistone proteins controls gene expression in a variety of cellular processes, with transcription being activated by acetyltransferases and silenced by deacetylases. We report here the formation and enzymatic characterization of a complex between the acetyltransferase p300 and histone deacetylases. The C/H3 region of p300 was found to co-purify deacetylase activity from nuclear cell extracts. A prototype of class I histone deacetylases, HDAC1, interacts with p300 C/H3 domain in vitro and in vivo. The p300-binding protein E1A competes with HDAC1 for C/H3 binding; and, like E1A, HDAC1 overexpression interferes with either activation of Gal4p300 fusion protein or p300-dependent co-activation of two C/H3-binding proteins, MyoD and p53. The exposure to deacetylase inhibitors could reverse the dominant-negative effect of a C/H3 fragment insulated from the rest of the molecule, on MyoD- and p53-dependent transcription, whereas inhibition by E1A was resistant to trichostatin A. These data support the hypothesis that association between acetyltransferases and deacetylases can control the expression of genes implicated in cellular growth and differentiation, and suggest that the dominant-negative effect of the p300 C/H3 fragment relies on deacetylase recruitment.

Thyroid hormone receptors (TRs) and members of the myocyte enhancer factor 2 (MEF2) family are involved in the regulation of muscle-specific gene expression during myogenesis. Physical interaction between these two factors is required to synergistically activate gene transcription. p300/cAMP-response-element-binding-protein ('CREB')-binding protein (CBP) interacting with transcription factors is able to increase their activity on target gene promoters. We investigated the role of p300 in regulating the TR—MEF2A complex. To this end, we mapped the regions of these proteins involved in physical interactions and we evaluated the expression of a chloramphenicol acetyltransferase (CAT) reporter gene in U2OS cells under control of the α-myosin heavy chain promoter containing the thyroid hormone response element (TRE). Our results suggested a role of p300/CBP in mediating the transactivation effects of the TR—retenoid X receptor (RxR)—MEF2A complex. Our findings showed that the same C-terminal portion of p300 binds the N-terminal domains of both TR and MEF2A, and our in vivo studies demonstrated that TR, MEF2A and p300 form a ternary complex. Moreover, by the use of CAT assays, we demonstrated that adenovirus E1A inhibits activation of transcription by TR—RxR—MEF2A—p300 but not by TR—RxR—MEF2A. Our data suggested that p300 can bind and modulate the activity of TR—RxR—MEF2A at TRE. In addition, it is speculated that p300 might modulate the activity of the TR—RxR—MEF2A complex by recruiting a hypothetical endogenous inhibitor which may act like adenovirus E1A.

We identified 220-kD protein in bovine skeletal muscle homogenate by affinity chromatography on an agarose column and subsequent SDS-PAGE. Peptide mass fingerprinting (MALDI mass spectrometry) and internal sequence analysis revealed that this protein has homology with several members of the myosin superfamily, particularly with human cardiac β-myosin heavy chain (β-MHC). A rabbit polyclonal antibody against the 220-kD protein specifically stained a 220-kD band in Western blots of skeletal muscle homogenate. Immunohistochemical experiments on cryostat sections demonstrated that in skeletal muscle this protein is exclusively localized at the neuromuscular junctions, no immunoreactivity being present at the myofibril level. Because of its relative homology with cardiac β-MHC, we also investigated the distribution of the 220-kD protein in bovine heart. In cardiac fibers, 220-kD protein-related immunoreactivity was restricted to the intercalated disks, whereas myofibrils were completely devoid of specific immunoreactivity. This distribution pattern was completely different from that of cardiac β-MHC, which involved myofibrils. Because of the above biochemical and immunohistochemical features, the 220-kD protein we have identified is suggested to be a novel member of the non-muscle (non-sarcomeric) myosin family.

Although the structural and pharmacological properties of endothelin (ET) receptors have been studied, little is known concerning their physiological significance, even if each subtype is supposed to have a distinct physiological action. Thus, to further elucidate the physiological function of this receptor, we examined the presence and distribution of the endothelin-B receptor (ET(B)) subtype in tissues of the lizard Podarcis sicula, using immunoblotting and immunohistochemistry. Immunoblotting indicated that, although the ET(B) receptor appears to be ubiquitous, it is present at different levels in the tissues examined. Furthermore, immunohistochemistry showed that this receptor is very abundant in endothelial cells of all tissues, suggesting that there is an ET(B)-mediated autocrine system of endothelin, which plays an important role in the regulation of endothelial cell function. On the other hand, the presence of ET(B) immunoreactivity also in endocrine systems such as adrenal and thyroid glands suggests an involvement also in the paracrine system of these organs.

The retinoblastoma family of proteins, pRb/p105, p107, and pRb2/ p130, cooperate to regulate cell cycle progression through the G1 phase of the cell cycle. Each of the family members realize their common goal of G1-S checkpoint regulation through overlapping and unique growth regulatory pathways. We took advantage of a tetracycline-regulated gene expression system to control the expression of RB2/p130 in JC virus-induced hamster brain tumor cells to study in vivo the molecular mechanisms used by pRb2/p130 to elicit its growth-suppressive function. We have previously used this system to demonstrate that induction of pRb/ p130 expression suppresses tumor growth in vivo by overcoming neoplastic transformation mediated by the large T-antigen oncoprotein of JCV (JCV TAg). Here we found that induction of pRb2/p130 in vivo specifically inhibits cyclin A- and cyclin E-associated kinase activity and by doing so induces p27Kip1 levels presumably by inhibiting p27Kip1-targeted proteolysis by cyclin E-Cdk2 phosphorylation of p27Kip1. RB2/p130 induction also decreased cyclin A and the transcription factor E2F-1 while increasing cyclin E at both the transcriptional and protein levels of expression. The growth inhibitory activity of pRb2/p130 also correlated with its E2F-binding capacity. Furthermore, p27Kip1 and pRb2/p130 were found to be targets of the JCV TAg oncoprotein and to interact in vivo with each other independently from the presence of TAg. Interestingly, pRb2/p130 expression negatively modulated the binding of p27Kip1 to JCV TAg. These data suggest that pRb2/p130 and p27Kip1 may cooperate in regulating cellular proliferation, and both may be involved in a negative feedback regulatory loop with cyclin E.

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse a- and b-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states. J. Cell. Biochem. 77:529-539, 2000. © 2000 Wiley-Liss, Inc.

An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using the yeast two-hybrid system. Further analysis restricted the epitope responsible for the binding within the 74 N-terminal amino acids of cyclin D3, independent of the LXCXE amino acid motif present in the D-type cyclin N-terminal region. In a coprecipitation assay in T98G cells, a human glioblastoma cell line, the C-terminal domain of pRb2/p130 was able to interact solely with cyclin D3, while the corresponding portion of pRb interacted with either cyclin D3 or cyclin D1. In T98G cells, endogenous cyclin D3-associated kinase activity showed a clear predisposition to phosphorylate preferen- tially the C-terminal domain of pRb2/p130, rather than that of pRb. This propensity was also confirmed in LAN-5 human neuroblastoma cells, where phosphorylation of the pRb2/p130 C-terminal domain and expression of cyclin D3 also decreased remarkably in the late neural differentiation stages. J. Cell. Biochem. 75:698-709, 1999. r 1999 Wiley-Liss, Inc.

We investigate the role played by dendritic cells (DCs) in the non-obese diabetic (NOD) mouse pancreas. The early peri-islet, nondestructive infiltration phase, and intra-islet, destructive infiltration phase, which immediately precedes overt diabetes, are studied. Results show that infiltrating cells are Ia-b, ICAM-1, and, mainly, MIDC-8 immunoreactive (ir). These data from silica-treated animals and ultrastructural observations strongly support the hypothesis that DCs are both Ia-b-ir and ICAM-1-ir and that they exert a pivotal role during the period of early infiltration. This is a novel finding for NOD mice and increases the interest for this protective cell type during the rather complex islet infiltration process. Moreover, the cytokine profile demonstrates that Th2 protective cytokines are specific for peri-islet infiltrate. Disappearance of DCs from the infiltrate is concomitant with both the formation of intra-islet infiltration and the increase in proinflammatory Th1 cytokine levels. This further supports the hypothesis that DCs may exert a protective role against diabetes development.

The inability of myocytes to reenter the cell cycle in vitro may result from a block in the activation of cyclins and cyclin-dependent kinases (cdk). This inhibition may not occur in vivo because myocyte proliferation is present in the failing heart. Thus, cardiac failure was induced by ventricular pacing in dogs, and changes in the quantity of cyclin D2, cyclin A, cyclin B, cdk2, and cell-division cycle-2 (cdc2) in control and paced myocytes were measured. The kinase activity of these nuclear proteins was also established. Finally, DNA synthesis and mitotic indices in myocytes were evaluated. Cyclin D2 in myocytes increased 7-fold after pacing, and cyclin D2-associated kinase activity increased 3-fold. Similarly, cyclin A quantity and activity increased 4-fold. Comparable changes were observed for cyclin B. cdc2 protein increased 8-fold, and cdk2 and cdc2 activity increased 3-fold and 5-fold, respectively. DNA synthesis was detected in 556 myocyte nuclei/10(6) and 2,467 myocyte nuclei/10(6) in control and paced hearts, respectively. Corresponding mitotic indices were 16/10(6) and 95/106, respectively. In conclusion, myocytes react to cardiac failure by activating cyclins and cdk, which are coupled with cell regeneration and the recovery of muscle mass.

CDK9 has been recently shown to have increased kinase activity in differentiated cells in culture and a differentiated tissue-specific expression in the developing mouse. In order to identify factors that contribute to CDK9's differentiation-specific function, we screened a mouse embryonic library in the yeast two-hybrid system and found a tumor necrosis factor signal transducer, TRAF2, to be an interacting protein. CDK9 interacts with a conserved domain in the TRAF-C region of TRAF2, a motif that is known to bind other kinases involved in TRAF-mediated signaling. Endogenous interaction between the two proteins appears to be specific to differentiated tissue. TRAF2-mediated signaling may incorporate additional kinases to signal cell survival in myotubes, a cell type that is severely affected in TRAF2 knockout mice.

The cdc2-family of serine/threonine kinases and their binding partners recently were implicated in developmental roles. We previously cloned a cdc2-related kinase, cdk9/PITALRE, that is able to phosphorylate the retinoblastoma protein in vitro. We describe here the cloning and the characterization of the mouse homolog of cdk9/PITALRE. The murine cDNA is 98% identical with humans and is expressed at high levels in brain and kidney tissues. The kinase activity and protein expression of cdk9/PITALRE were highest in terminally differentiated tissues such as the muscle and brain. In situ immunohistology and immunofluorescence detected cdk9/PITALRE protein not only within terminally differentiated cells such as muscle and neuronal cells, but also in proliferating cells. C2C12 and P19 cells induced to differentiate along muscle and neural lineages peaked in cdk9/PITALRE kinase activity at the end of differentiation. These results suggest that, among other roles, cdk9/PITALRE plays a role not unlike cdk5 in the differentiation of certain cell types. J. Cell. Physiol. 177:206–213, 1998. © 1998 Wiley-Liss, Inc.

The histone H4 gene promoter provides a paradigm for defining transcriptional control operative at the G1/S phase transition point in the cell cycle. Transcription of the cell cycle-dependent histone H4 gene is upregulated at the onset of S phase, and the cell cycle control element that mediates this activation has been functionally mapped to a proximal promoter domain designated Site II. Activity of Site II is regulated by an E2F-independent mechanism involving binding of the oncoprotein IRF2 and the multisubunit protein HiNF-D, which contains the homeodomain CDP/cut, CDC2, cyclin A, and the tumor suppressor pRb. To address mechanisms that define interactions of Site II regulatory factors with this cell cycle control element, we have investigated these determinants of transcriptional regulation at the G1/S phase transition in FDC-P1 hematopoietic progenitor cells. The representation and activities of histone gene regulatory factors were examined as a function of FDC-P1 growth stimulation. We find striking differences in expression of the pRb-related growth regulatory proteins (pRb/p105, pRb2/p130, and p107) following the onset of proliferation. pRb2/p130 is present at elevated levels in quiescent cells and declines following growth stimulation. By contrast, pRb and p107 are minimally represented in quiescent FDC-P1 cells but are upregulated at the G1/S phase transition point. We also observe a dramatic upregulation of the cellular levels of pRb2/p130-associated protein kinase activity when S phase is initiated. Selective interactions of pRb and p107 with CDP/cut are observed during the FDC-P1 cell cycle and suggest functional linkage to competency for DNA binding and/or transcriptional activity. These results are particularly significant in the context of hematopoietic differentiation where stringent control of the cell cycle program is requisite for expanding the stem cell population during development and tissue renewal. J. Cell. Biochem. 66:512–523, 1997. © 1997 Wiley-Liss, Inc.

The Cdk2 kinase has long been known to be involved in the progression of mammalian cells past the G1 phase restriction point and through DNA replication in the cell cycle. The Rb family of proteins, consisting of pRb, p107, and pRb2/p130, has also been shown to monitor progression of G1 phase, mostly through their interaction with E2F family members. p107 is able to inhibit Cdk2 kinase activity through this interaction via a p21-related domain present in the C terminus of the protein. We show here that pRb2/p130 also possesses this activity, but through a separate domain. Moreover, we correlate the increased expression of pRb2/p130 during various cellular processes with the decreased kinase activity of Cdk2. We hypothesize that pRb2/p130 may act not only to bind and modify E2F activity, but also to inhibit Cdk2 kinase activity in concert with p21 in a manner different from p107.

Transgenic mice harboring the early genome from the human neurotropic JC virus, JCV, develop massive abdominal tumors of neural crest origin during 6–8 months after birth and succumb to death a few weeks later. The viral early protein, T-antigen, which possesses the ability to transform cells of neural origin, is highly expressed in the tumor cells. Immunoblot analysis of protein extract from tumor tissue shows high level expression of the tumor suppressor protein, p53, in complex with T-antigen. Expression of p21, a downstream target for p53, which controls cell cycle progression by regulating the activity of cyclins and their associated kinases during the G1 phase, is extremely low in the tumor cells. Whereas the level of expression and activity of cyclin D1 and its associated kinase, cdk6, was modest in tumor cells, both cyclin A and E, and their kinase partners, cdk2 and cdk4, were highly expressed and exhibited significant kinase activity. The retinoblastoma gene product, pRb, which upon phosphorylation by cyclins:cdk induces rapid cell proliferation, was found in the phosphorylated state in tumor cell extracts, and was detected in association with JCV T-antigen. The transcription factor, E2F-1, which dissociates from the pRb–E2F-1 complex and stimulates S phase-specific genes upon phosphorylation of pRb and/or complexation of pRb with the viral transforming protein, was highly expressed in tumor cells. Accordingly, high level expression of the E2F-1-responsive gene, proliferating cell nuclear antigen (PCNA), was detected in the tumor cells. These observations suggest a potential regulating pathway that, upon expression of JCV T-antigen, induces formation and progression of tumors of neural origin in a whole animal system. J Cell. Biochem. 67:223–230, 1997. © 1997 Wiley-Liss, Inc.

The retinoblastoma gene family is composed of three members: the retinoblastoma gene, one of the most well studied tumor suppressor genes and two related proteins, p107 and pRb2lp130. These three proteins share many structural and functional features and play a fundamental role in growth control. Intense investieation of these moteins has identified a series of U similar cell-cycle regulators and transcription factors with which they interact. Although the precise function of the retinoblastoma gene product and its relatives remains unknown, recent data suggests that they play parallel roles in controlling cell cycle progression and promoting cellular differentiation. In this review, we will attempt to clarify some of the molecular mechanisms by which these three related proteins cooperate to control cellular proliferation and differentiation.

ets-2 is a member of a family of transcription factors implicated in the regulation of gene expression during cell proliferation, cell differentiation, and development. We report that the ets-2 protein transactivates the promoter of the cdc2 gene which encodes a 34-kDa serine-threonine kinase required for mitotic initiation in mammalian cells. Transactivation occurs via specific interaction with multiple ets binding sites in the 5′ flanking region of the gene. In BALB/c3T3 rodent fibroblasts constitutively expressing ets-2 and cultured in either 10 or 0.5% serum, cdc2 expression and its associated histone H1 kinase activity are increased, compared to control cells. Such increased activity correlates with elevated levels of cyclin A but not cyclin B1. Furthermore, ets-2-transfected, but not parental, BALB/c3T3 cells, grow under low serum conditions, albeit at a reduced rate. These data demonstrate that ets-2 plays a direct role in the regulation of cdc2 expression and raise the possibility that ets-2 participates in the coordinated regulation of cdc2 cyclin A expression which is essential for the modulation of cdc2-regulated processes.

The cell division cycle have been shown to be regulated by a closely-related family of protein kinases named CDKs (by cyclin-dependent kinases). Using a PCR-based cloning technique, we have isolated cDNAs encoding a human CDC2-related protein kinase. The full-length cDNA accommodates an open reading frame that does not contain any ATG initiation codon upstream of the sequence encoding the catalytic domain of this putative kinase. Three putative non-ATG initiation codons have been detected. Starting at the most 5' non-ATG initiation site, the encoded product is 316 amino acids long with a predicted molecular weight of 35.8 kDa. Analysis of the deduced amino acid sequence showed it to contain the XI subdomains present in all known protein kinases and a PSTAIRE-like motive, PISSLRE, which temporarily names this kinase. PISSLRE is most related to p58/GTA (55% identity in the catalytic domain), the galactosyl transferase associated protein, which has been shown to inhibit entry into S-phase when over-expressed in CHO cells. PISSLRE shares 38-45% identity with all CDKs and contains the regulatory Tyr and Thr residues present in most of the members of the CDK family of protein kinases, which suggests similar modes of regulation. PISSLRE is expressed in all human tissues tested, including those which contain high proportion of terminally differentiated cells. However, the levels of the PISSLRE transcripts are dissimilar among different tissues.

The promoter of the Insulin-like growth factor I (IGF-I) gene is activated by the Simian Virus 40 large T antigen (SVLT), and one of the elements responding to SVLT activation has been localized to a short 124 bp immediately upstream of the first initiation of transcription site. This short promoter contains an E2F binding site, that, in gel shifts, binds a protein complex, but only when the promoter activity is reduced or absent. A mutation in the E2F binding site deregulates the activity of the promoter, which becomes active even in those conditions in which the wild type promoter is inactive. By using antibodies in gel retardation analyses, we can show that the different protein complexes include, at least, the following proteins: E2F, cyclin A and p107. We conclude that the short IGF-I promoter is negatively regulated by an E2F binding site that complexes with several proteins. Our data suggest that disaggregation of these complexes by the action of SVLT (or other activators) increases expression from the promoter, thus establishing a link between the regulation of cell proliferation by growth factors and the E2F-associated proteins.

By means of the yeast two-hybrid system, we have discovered a novel physical interaction between the adenovirus E1A oncoprotein and Ran, a small GTPase which regulates nucleocytoplasmic transport, cell cycle progression, and mitotic spindle organization. Expression of E1A elicits induction of S phase and centrosome amplification in a variety of rodent cell lines. The induction of supernumerary centrosomes requires functional RCC1, the nucleotide exchange factor for Ran and, hence, a functional Ran network. The E1A portion responsible for the interaction with Ran is the extreme NH(2)-terminal region (amino acids 1-36), which is also required for the induction of centrosome amplification. In an in vitro assay with recombinant proteins, wild-type E1A interferes with nucleotide exchange on Ran, whereas an E1A mutant, deleted from the extreme NH(2)-terminal region, does not. In addition, we detected an in vitro interaction between Ran and HPV-16 E7 and SV40 large T antigen, two oncoproteins functionally related to E1A. These findings suggest a common pathway of these oncoproteins in eliciting virus-induced genomic instability.

The Rb (retinoblastoma) gene family is composed of three members: the RE gene (one of the best-studied tumor suppressor genes) and two related genes, p107 and pRb2/ p130. These three proteins share many structural and functional features and play a fundamental role in growth control. Using immunocytochemical techniques, we evaluated a variety of lung tumor specimens for the expression of this family of proteins and compared protein expression with the histological grading of the tumors and with the expression of the proliferating cell nuclear antigen. These Rb family members displayed distinctive patterns when compared and contrasted using different parameters. The highest percentage of undetectable levels in all of the specimens examined and the tightest inverse correlation (P) with the histological grading and with proliferating cell nuclear antigen expression in the most aggressive tumor types were found for pRb2/p130, which may suggest an important role for this protein in the pathogenesis and progression of lung cancer.