Sgk1 enhances RANBP1 transcript levels and decreases taxol sensitivity in RKO colon carcinoma cells.

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of epithelial sodium channel-mediated sodium transport and is involved in the transduction of growth factor-dependent cell survival and proliferation signals. Growing evidence now points to Sgk1 as a key element in the development and/or progression of human cancer. To gain insight into the mechanisms through which Sgk1 regulates cell proliferation, we adopted a proteomic approach to identify up- or downregulated proteins after Sgk1-specific RNA silencing. Among several proteins, the abundance of which was found to be up- or downregulated upon Sgk1 silencing, we focused our attention of RAN-binding protein 1 (RANBP1), a major effector of the GTPase RAN. We report that Sgk1-dependent regulation of RANBP1 has functional consequences on both mitotic microtubule activity and taxol sensitivity of cancer cells.

A small molecule based on the pRb2/p130 spacer domain leads to inhibition of cdk2 activity, cell cycle arrest and tumor growth reduction in vivo.

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.

Retinoblastoma family proteins as key targets of the small DNA virus oncoproteins.

RB, the most investigated tumor suppressor gene, is the founder of the RB family of growth/tumor suppressors, which comprises also p107 (RBL1) and Rb2/p130 (RBL2). The protein products of these genes, pRb, p107 and pRb2/p130, respectively, are also known as 'pocket proteins', because they share a 'pocket' domain responsible for most of the functional interactions characterizing the activity of this family of cellular factors. The interest in these genes and proteins springs essentially from their ability to regulate negatively cell cycle processes and for their ability to slow down or abrogate neoplastic growth. The pocket domain of the RB family proteins is dramatically hampered in its functions by the interference of a number of proteins produced by the small DNA viruses. In the last two decades, the 'viral hypothesis' of cancer has received a considerable renewed impulse from the notion that small DNA viruses, such as Adenovirus, Human papillomavirus (HPV) and Polyomavirus, produce factors that can physically interact with major cellular regulators and alter their function. These viral proteins (oncoproteins) act as multifaceted molecular devices that have evolved to perform very specific tasks. Owing to these features, viral oncoproteins have been widely employed as invaluable experimental tools for the identification of several key families of regulators, particularly of the cell cycle homeostasis. Adenovirus early-region 1A (E1A) is the most widely investigated small DNA tumor virus oncoprotein, but relevant interest in human oncology is raised by the E1A-related E7 protein from transforming HPV strains and by Polyomavirus oncoproteins, particularly large and small T antigens from Simian virus 40, JC virus and BK virus.

Expression of p21 in SV40 large T antigen positive human pleural mesothelioma: relationship with survival.

BACKGROUND: Mesothelioma is the most commonly occurring primary pleural neoplasm. Several studies have documented an increase in the incidence of this malignancy during the last decades. Although the association between asbestos exposure and development of mesothelioma is generally accepted, the exact mechanism of carcinogenesis is unknown. Recently, Simian virus 40 large T antigen (SV40 Tag) expression has been detected in pleural mesothelioma. The ability of SV40 oncoproteins to inactivate p53 and retinoblastoma tumour suppressor proteins has been proposed as an important step in the pathogenesis of human mesothelioma. METHODS: To obtain a better understanding of the molecular mechanisms of the pathogenesis of mesothelioma, the expression of the cell cycle inhibitor p21(WAF1/CIP1) (p21), a downstream target of p53, was evaluated immunohistochemically in a group of 29 mesothelioma specimens already characterised for the presence of SV40 Tag sequences. RESULTS: Statistical analysis did not reveal any correlation between p21 expression and histopathological type of mesothelioma using the kappa(2) test (p=0.577). A significant positive relationship was found between p21 expression level and the patients' overall survival according to the Kaplan-Meier survival curves and using a log rank test (median difference in survival 7 months, 95% CI 4.8 to 9.9; p<0.001). CONCLUSIONS: Determination of p21 expression bears a prognostic significance in patients affected with mesothelioma, further underlining the role of SV40 in the pathogenesis of malignant pleural mesothelioma.

Expression of AP-2 transcription factor and of its downstream target genes c-kit, E-cadherin and p21 in human cutaneous melanoma.

The AP-2 transcription factor plays a pivotal role in regulating the expression of several genes involved in tumor growth and progression of melanoma. We determined, by Western blot, variation in the level of expression of AP-2 and three of its downstream targets, c-kit, E-cadherin, and p21 in several human melanoma cell lines and, by immunohistochemistry, in a group of 99 histological samples including benign and malignant melanocytic lesions. A significant negative correlation between AP-2 expression level and tumor thickness was found. Moreover, AP-2 expression was positively associated with E-cadherin and c-kit expression. In contrast, there was a significant negative association between AP-2 and p21 expression levels. These findings suggest that p21 is independent of AP-2 transactivator function during the latest phases of melanoma progression. Finally, AP-2, c-kit, E-cadherin, and p21 expression levels did not show to be able to distinguish between dysplastic nevi and nevi without dysplasia. We conclude that changes in the expression of these proteins are involved in the later phases of melanoma progression, and may be responsible for the transition from local invasive melanoma to metastasis.

Retinoblastoma family proteins induce differentiation and regulate B-myb expression in neuroblastoma cells.

BACKGROUND: The expression of several genes is modulated during neuroblastoma differentiation. The retinoblastoma family proteins, pRb, p107 and pRb2/p130, act in the repression of proliferation genes, interacting mainly with the E2F transcription factors. PROCEDURE AND RESULTS: In this study, we found that, in neuroblastoma cell lines, pRb and p107 proteins decreased, undergoing progressive dephosphorylation, whereas pRb2/p130 increased at late stages of differentiation. B-myb expression was down-regulated in association with the up-regulation of pRb2/p130, the major partner of E2F on the E2F site of the B-myb promoter in differentiated cells. Transfection of each of the retinoblastoma family genes in neuroblastoma cells was able to induce neural differentiation, to inhibit 3H-thymidine incorporation, and to down-regulate B-myb promoter activity. CONCLUSIONS: In conclusion, our data suggest a major contribution of retinoblastoma proteins, and especially of pRb2/p130, in B-myb promoter regulation and demonstrate the induction of neural differentiation by p107 and pRb2/p130, suggesting a role of these proteins in triggering differentiation-specific genes.

Who is the boss in the retinoblastoma family? The point of view of Rb2/p130, the little brother.

This review portrays an updated overview about the possible tumor suppressive properties of the Rb2/p130 gene, the third member of the retinoblastoma (RB) family of genes, including RB itself and p107. After a brief analysis of the established structural and functional similarities among the three genes, the main purpose is to critically analyze present evidence whether Rb2/p130 shares the role of a tumor suppressor. Taking into account the well-proven growth suppressive properties of Rb2/p130 and p107, we discuss the analysis of mutated or deleted forms of Rb2/p130 found in a number of human cancers. Finally, we take into consideration the data provided by the targeted disruption of each RB family gene, alone or in combination, in the mouse model.

Interplay between the antimetastatic nm23 and the retinoblastoma-related Rb2/p130 genes in promoting neuronal differentiation of PC12 cells.

Increasing evidence indicates that the nm23 genes, initially documented as suppressors of metastasis progression, are involved in normal development and differentiation. We have shown previously that the murine nm23 gene enhances pheochromocytoma PC12 cells responsiveness to NGF by accelerating cell growth arrest and neurite outgrowth. The present study was aimed at elucidating the mechanisms by which nm23 controls cell proliferation and promotes neuronal differentiation. We demonstrated that nm23 modulates the expression of the Rb2/p130 gene, a negative regulator of cell cycle progression also implicated in the maintenance of the differentiated state. Furthermore, we showed that nm23-H1 mutants, defective in inhibiting the invasive phenotype, downregulate Rb2/p130 expression and inhibit NGF-induced PC12 cell differentiation. In synthesis, our results provide first evidence of interplay between the nm23 and the Rb2/p130 genes in driving PC12 cells neuronal differentiation and suggest that the antimetastatic and the differentiative nm23 functions can have similar features.

The retinoblastoma-related Rb2/p130 gene is an effector downstream of AP-2 during neural differentiation.

Rb2/p130, a member of the Retinoblastoma family of growth and tumour suppressor genes, is extensively implicated in the control of cell cycle and differentiation. The minimal promoter region of Rb2/p130 in T98G human glioblastoma cells was identified and its analysis revealed the presence of a KER1 palindromic sequence able to bind the transcription factor AP-2, a regulatory protein that plays a crucial role in ectodermal differentiation. This KER1 site interacted in vitro with AP-2, and AP-2 overexpression increased Rb2/p130 transcription and translation. We also found that rat PC12 pheochromocytoma cells, when induced to differentiate by NGF, displayed an increase of AP-2 protein levels and of Rb2/p130 transcription and protein levels. AP-2-transfected PC12 cells displayed enhanced transcription and translation of Rb2/p130 and of the cdk inhibitor p21(WAF1/CIP1), a gene known to be under the control of AP-2, but unable by itself to elicit PC12 differentiation. Overexpression of either AP-2 or Rb2/p130 elicited per se cell differentiation in the absence of NGF, while coexpression of AP-2B, a negative regulator of AP-2 transcriptional activity, inhibited only AP-2-induced differentiation. Altogether, these results indicate that Rb2/p130 is a critical effector of AP-2 in sustaining ectodermal differentiation.

Pattern of expression of cyclin T1 in human tissues.

Cyclin T1 was recently identified, together with cdk9 (previously named PITALRE), as part of the TAK multiprotein complex, a co-factor targeted by the human immunodeficiency virus Type 1 (HIV-1) protein named Tat, suggesting a role for this complex in transcription elongation. Although studies on mRNA and protein expression have shown that cyclin T1 is ubiquitous in adult human tissues, no data have yet been reported regarding the expression of this protein in different cell lineages. Using a polyclonal antiserum raised against cyclin T1, we investigated the pattern of expression of this protein in adult human tissues by immunohistochemistry. Cyclin T1 was expressed ubiquitously, although different levels of expression were found in various organs. Some specialized tissues, such as blood, lymphoid tissues, and cells of connective tissue origin, showed high cyclin T1 expression. These specific expression patterns are only partially justified by some well-known specialized functions of cyclin T1 in certain cell types, such as its involvement in peripheral blood lymphocytes and monocyte differentiation. The high expression level found in other tissues suggests new possible roles for cyclin T1 in cell types other than those of lymphoid tissue.

Cyclin T2a gene maps on human chromosome 2q21.

Cyclin T2a was recently identified as one of the regulatory subunits of the cdk-cyclin complex P-TEFb, the most studied positive factor in the regulation of transcription elongation. By fluorescent in situ hybridization (FISH), the gene codifying for cyclin T2a has been mapped on human chromosome 2q21. This locus also has been linked to different forms of myopathy. By use of a new specific antiserum raised against cyclin T2a, the immunohistochemical pattern of expression of cyclin T2a in human tissues has been examined and compared to that of cyclin T1, described in the previous report. The observation that immunohistochemical expression of cyclin T2a was high in skeletal muscle cells, whereas it was undetectable in two cases of centronuclear myopathy, together with its chromosomal location, suggests an involvement of the cdk9-cyclin T2a complex in this disease.

RACK1 interacts with E1A and rescues E1A-induced yeast growth inhibition and mammalian cell apoptosis.

The adenoviral E1A proteins are able to promote proliferation and transformation, inhibit differentiation, induce apoptosis, and suppress tumor growth. The extreme N terminus and conserved region one of E1A, which are indispensable for transcriptional regulation and for binding to p300/CBP, TBP, and pCAF, play essential roles in these abilities. These observations strongly suggest an intrinsic link between E1A-mediated transcriptional regulation and other effects. In this report, we show that E1A inhibits the normal growth of Saccharomyces cerevisiae HF7c, and this inhibition also depends on the domains required for transcriptional regulation. We demonstrate that E1A associates with histone acetyltransferase activity and represses the transactivation activity of transcription factor in S. cerevisiae, suggesting that E1A may suppress the expression of genes required for normal growth. Based on yeast growth rescue, we present a genetic screening strategy that identified RACK1 as an E1A antagonizing factor. Expression of human RACK1 efficiently relieves E1A-mediated growth inhibition in HF7c and protects human tumor cells from E1A-induced apoptosis. Finally, we show that RACK1 decreases E1A-associated histone acetyltransferase activity in yeast and mammalian cells, and physically interacts with E1A. Our data demonstrate that RACK1 is a repressor of E1A, possibly by antagonizing the effects of E1A on host gene transcription.

A serine 37 mutation associated with two missense mutations at highly conserved regions of p53 affect pro-apoptotic genes expression in a T-lymphoblastoid drug resistant cell line.

The p53 protein accumulates rapidly through post-transcriptional mechanisms following cellular exposure to DNA damaging agents and is also activated as a transcription factor leading to growth arrest or apoptosis. Phosphorylation of p53 occurs after DNA damage thereby modulating its activity and impeding the interaction of p53 with its negative regulator oncogene Mdm2. The serines 15 and 37 present in the amino terminal region of p53 are phosphorylated by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. In order to verify if specific p53 mutations occur in the multi-drug resistance phenotype, we analysed the p53 gene in two T-lymphoblastoid cell lines, CCRF-CEM and its multi-drug-resistant clone CCRF-CEM VLB100, selected for resistance to vinblastine sulfate and cross-resistant to other cytotoxic drugs. Both cell lines showed two heterozygous mutations in the DNA binding domain at codons 175 and 248. The multi-drug resistant cell line, CCRF-CEM VLB100, showed an additional mutation that involves the serine 37 whose phosphorylation is important to modulate the protein activity in response to DNA damage. The effects of these mutations on p53 transactivation capacity were evaluated. The activity of p53 on pro-apoptotic genes expression in response to DNA damage induced by (-irradiation, was affected in the vinblastine (VLB) resistant cell line but not in CCRF-CEM sensitive cell line resulting in a much reduced apoptotic cell death of the multi-drug resistant cells.

nm23: unraveling its biological function in cell differentiation.

Tumor suppressor genes have a pivotal role in normal cells regulating cell cycle processes negatively. Furthermore, the inhibition of cell proliferation is a crucial step in the achievement of cell differentiation. Increasing evidence suggests that the nm23 genes, initially documented as suppressors of the invasive phenotype in some cancer types, are involved in the control of normal development and differentiation. In this review, we summarize some data concerning the involvement of the nm23 genes in development and differentiation, attempting to delineate an overall view of many facets of their biological role.

Interaction between the pRb2/p130 C-terminal domain and the N-terminal portion of cyclin D3.

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 preferentially 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.

Enhancement of doxorubicin content by the antitumor drug lonidamine in resistant Ehrlich ascites tumor cells through modulation of energy metabolism.

The effect of the antitumor drug lonidamine (LND) on respiration, aerobic glycolysis, adenylate pool, doxorubicin (DOX) uptake, and efflux in DOX-resistant and DOX-sensitive Ehrlich tumor cells was investigated. The results may be summarized as follows: 1) In both types of cells, LND inhibited both respiration and glycolysis in a dose-dependent manner and lowered the ATP concentration. The effect was more marked in cells incubated in glucose-free medium; 2) LND raised, to a remarkable extent, the intracellular content of DOX in resistant and sensitive cells respiring on endogenous substrates because of reduced ATP availability, whereas in glucose-supplemented medium, where both respiration and glycolysis contributed to ATP synthesis, the increase was lower; and 3) when LND was added to DOX-loaded cells, it failed to significantly inhibit DOX efflux because of time-dependent phenomena. These findings indicated that LND, a drug currently employed in tumor therapy, might also be useful in reducing or overcoming multidrug resistance (MDR) of those cells with a reduced ability to accumulate and retain antitumor drugs.

The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation.

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.

Retinoblastoma-related protein pRb2/p130 and its binding to the B-myb promoter increase during human neuroblastoma differentiation.

Neuroblastoma cells can undergo neural differentiation upon treatment with a variety of chemical inducers and growth factors. During this process, many cell cycle-related genes are downregulated while differentiation-specific genes are triggered. The retinoblastoma family proteins, pRb, p107, and pRb2/p130, are involved in transcriptional repression of proliferation genes, mainly through their interaction with the E2F transcription factors. We report that pRb2/p130 expression levels increased during differentiation of neuroblastoma cell line LAN-5. On the other hand, both pRb and p107 decreased and underwent progressive dephosphorylation at late differentiation times. The expression of B-myb and c-myb, two targets of the retinoblastoma family proteins, were downregulated in association with the increase of pRb2/p130, which was detected as the major component of the complex with E2F on the E2F site of the B-myb promoter in differentiated cells. Interestingly, E2F4, a preferential partner of p107 and pRb2/p130, was upregulated and underwent changes in cellular localization during differentiation. In conclusion, our data suggest a major role of pRb2/p130 in the regulation of B-myb promoter during neural differentiation despite the importance of cofactors in modulating the function of the retinoblastoma family proteins.

Effect of bcr-abl oligodeoxynucleotides on the clonogenic growth of chronic myelogenous leukaemia cells.

We studied the effect of phosphorothioate oligodeoxynucleotides ([S]ODNs) complementary to the bcr-abl junction on cells taken at diagnosis from 41 patients with Philadelphia-positive chronic myelogenous leukaemia (CML). Experiments included the evaluation of the anti-leukaemic effect of 16- and 26-mer antisense [S]ODNs on both mononuclear and CD34+ cells, evaluation of incubation time and correlation of colony growth inhibition with the down-regulation of p210(bcr-abl). At the same time, the uptake of [S]ODNs by mononuclear and purified CD34+ cell populations and the cross-hybridization of 26- and 16-mer [S]ODNs with the complementary sequences were evaluated. After incubation for 120 h with 26-mer antisense [S]ODNs on mononuclear cells, overall mean colony recovery was 41.9% of the untreated control samples; in particular, a significant reduction in colony formation was observed in 22 of the 35 cases tested. The effect of 26-mer ODNs on CD34+ cells was comparable to that observed on mononuclear cells in terms of colony inhibition; however, a higher proportion of cases showed a significant inhibition of colony formation. In comparison with the 26-mer antisense [S]ODNs, the anti-leukaemic effect of the 16-mer antisense [S]ODNs was less evident on mononuclear cells and comparable on CD34+ cells; however, a more specific effect was evident on both target cells. Hybridization experiments confirmed a partial cross-reactivity when the 26-mer ODNs were hybridized with their complementary sequence; this did not occur when 16-mer ODNs were similarly tested. Experiments aimed at evaluating the effect of the incubation time showed a significant increase in anti-leukaemic effect after a 120 h incubation period compared to that measured after a 24 h incubation period; this was parallelled by a progressive increase in the intracellular concentrations of [S]ODNs from day 1 to day 5. The accumulation of [S]ODNs correlated with a marked down-regulation of p210(bcr-abl) levels which was first detectable after 72 h of treatment. The down-regulation of p210(bcr-abl) levels following treatment with [S]ODNs showed a correlation between the effect of antisense [S]ODNs on leukaemic colony formation and protein expression. These studies confirm that, under optimal conditions of target cell culture and ODN size, antisense [S]ODNs complementary to the bcr-abl junction have specific anti-leukaemic effects.