Retinoblastoma tumor-suppressor protein phosphorylation and inactivation depend on direct interaction with Pin1.

Inactivation of the retinoblastoma protein (pRb) by phosphorylation triggers uncontrolled cell proliferation. Accordingly, activation of cyclin-dependent kinase (CDK)/cyclin complexes or downregulation of CDK inhibitors appears as a common event in human cancer. Here we show that Pin1 (protein interacting with NIMA (never in mitosis A)-1), a peptidylprolyl isomerase involved in the control of protein phosphorylation, is an essential mediator for inactivation of the pRb. Our results indicate that Pin1 controls cell proliferation by altering pRb phosphorylation without affecting CDK and protein phosphatase 1 and 2 activity. We demonstrated that Pin1 regulates tumor cell proliferation through direct interaction with the spacer domain of the pRb protein, and allows the interaction between CDK/cyclin complexes and pRb in mid/late G1. Phosphorylation of pRb Ser 608/612 is the crucial motif for Pin1 binding. We propose that Pin1 selectively boosts the switch from hypo- to hyper-phosphorylation of pRb in tumor cells. In addition, we demonstrate that the CDK pathway is responsible for the interaction of Pin1 and pRb. Prospectively, our findings therefore suggest that the synergism among CDK and Pin1 inhibitors holds great promise for targeted pharmacological treatment of cancer patients, with the possibility of reaching high effectiveness at tolerated doses.

The BRG1 ATPase of chromatin remodeling complexes is involved in modulation of mesenchymal stem cell senescence through RB-P53 pathways.

We focused our attention on brahma-related gene 1 (BRG1), the ATPase subunit of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, and analyzed its role in mesenchymal stem cell (MSC) biology. We hypothesized that deviation from the correct concentration of these proteins, which act at the highest level of gene regulation, may be deleterious for cells. We wanted to know what would happen if a cell had to cope with altered regulation of gene expression, either by upregulation or downregulation of BRG1. We assumed that cells would try to restore homeostasis or, alternatively, that the event could trigger senescence/apoptosis phenomena. To this end, in MSCs, we silenced BRG1gene. Knockdown of BRG1 expression induced a significant increase in senescent cells and decrease in apoptotic cells. It is interesting that BRG1 downregulation also induced an increase in heterochromatin. At the molecular level, these phenomena were associated with activation of retinoblastoma-like protein 2 (RB2)/P130- and P53-related pathways. Senescence was accompanied by reduced expression of some stemness-related genes. This is consistent with our previous research, which showed that BRG1 upregulation by ectopic expression also induced senescence processes. Together, these data suggest that BRG1 belongs to a class of genes whose expression is tightly regulated; hence, subtle alterations in BRG1 activity seem to negatively affect mechanisms regulating chromatin status and, in turn, impair cellular physiology.

Viral infections as a cause of cancer (review).

In order to promote carcinogenesis multiple factors must be orchestrated. The alteration of the cellular genome after a carcinogenic exposure may result in malignancy if apoptosis is prevented and the immune surveillance fails to eliminate the transformed cell. Infectious agents may exert these properties and transform a host cell. Viruses associated with human cancer are known as 'tumor viruses'. Most of them are capable of integrating into the host genome and have the ability to immortalize the target cell in order to allow their own replication. The infected cell expresses the viral genes, which are able to induce cell growth, proliferation and prevent apoptosis. This review focuses on Epstein-Barr virus, human papilloma virus, hepatitis C virus, hepatitis B virus, human herpes virus 8 and human T-cell leukemia virus, since they have been already established as causative agents of human cancer. An understanding of the viral replication mechanism may provide new targets for the development of specified viral therapy that may have an impact not only on viral infections but in human cancer as well.

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.

Study of viral integration of HPV-16 in young patients with LSIL.

AIMS: To investigate the physical status of human papillomavirus 16 (HPV-16) in low grade squamous intraepithelial lesions (LSILs) as a means of determining the percentage of viral integration. METHODS: Ninety two LSIL/HPV positive Thin Prep(TM) samples were initially tested for the E6 gene by the polymerase chain reaction (PCR) to identify the HPV-16 virus. To avoid false positive results, the specificity of the bands obtained from PCR was confirmed by Southern blot hybridisation with internal oligonucleotide probes. Next, a PCR screen for the E2 gene was performed to identify those samples in which the virus was integrated. Viral integration was detected in just over half of them. RESULTS: Twenty of the 92 samples were HPV-16 positive, as shown by PCR for the E6 gene. Southern blot analysis confirmed that 13 of these samples were positive for the viral E6 gene. Thus, viral integration was detected in just over a half of the samples positive for HPV-16. CONCLUSIONS: These data show that HPV-16 integration occurs in a subset of LSILs. The measurement of HPV-16 integration would be a helpful complementary tool for cytological evaluation in primary cervical screening to identify those patients at risk of developing high grade squamous intraepithelial lesions and cervical cancer.

Cyclin E and chromosome instability in colorectal cancer cell lines.

AIMS/BACKGROUND: The development of colorectal cancer depends on at least two distinct pathways involving genetic instability, namely: chromosome instability (CIN) and microsatellite instability. Cyclin E is involved in aneuploidy and several cancer types show an abnormal number of chromosomes. METHODS: Cyclin E protein and mRNA values were analysed in human fetal skin fibroblasts and five colorectal cancer cell lines. RESULTS: Cells with an aberrant number of chromosomes had higher cyclin E mRNA values and a significant increase in protein concentrations. CONCLUSIONS: These data suggest that cyclin E regulation is altered in aneuploid cells and is an important factor in the CIN pathway.

Genomic organization, promoter analysis, and chromosomal mapping of the mouse gene encoding Cdk9.

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.

Physical interaction between CDK9 and B-Myb results in suppression of B-Myb gene autoregulation.

B-Myb is a transcription factor belonging to the myb family, whose activity has been associated with augmented DNA synthesis and cell cycle progression. We showed recently that B-Myb autoregulates its own expression through promoter transactivation. We report in this study that CDK9, the cyclin T associated kinase, which phosphorylates and activates RNA-Polymerase II, suppresses B-Myb autoregulation through direct interaction with the carboxyl-terminus of the B-Myb protein. Down-regulation of the transactivating ability of B-Myb is independent of the kinase activity of CDK9, because a kinase deficient mutant (dn-CDK9) also represses B-myb gene autoregulation. Overexpression of CDK9 did not result in suppression of p53-dependent transactivation or inhibition of the basal activity of the promoters tested so far, demonstrating that CDK9 is a B-Myb-specific repressor. Rather, transfection of the dominant negative dn-CDK9 construct inhibited the basal activity of the reporter genes, confirming an essential role for CDK9 in gene transcription. In addition, Cyclin T1 restores B-Myb transactivating activity when co-transfected along with CDK9, suggesting that the down-regulatory effect observed on B-Myb is specifically due to CDK9 alone. Thus, our data suggest that CDK9 is involved in the negative regulation of activated transcription mediated by certain transcription factors, such as B-Myb. This may indicate the existence of a feedback loop, mediated by the different activities of CDK9, which links basal with activated transcription.

Independent prognostic role of p16 expression in lung cancer.

OBJECTIVES: The cyclin-dependent kinase p16 (also known as Ink4A, Mts1, Cdkn2, and Cdkn4i) has been proposed as a tumor suppressor gene mapped on chromosome segment 9p21. This study evaluated p16 protein expression in 135 lung cancer specimens and investigated potential genetic alterations occurring in this gene. RESULTS: We found altered p16 immunohistochemical expression to be a frequent event in lung cancer and to be independent of either the histologic type or any other clinical-pathologic feature. Western blot analyses performed on about one third of the specimens correlated highly with these results. In addition, we found p16 immunohistochemical expression to be a favorable prognostic factor in lung cancer in that its reduction or loss correlated with a worse outcome for the patients. Polymerase chain reaction amplification and direct sequencing of p16 exons 1 and 2 revealed no mutations, indicating that p16-altered expression in lung cancer is not necessarily linked to mutational events of these genes. CONCLUSIONS: We conclude that p16-altered expression is both an independent and frequent event in lung cancer and may have an important role in tumorigenesis and in malignant progression of a significant proportion of these cancers. However, the actual incidence and relevance of p16 mutations in this neoplasm continues to be debated, and its analysis seems inconclusive. Our results suggest a prognostic role for the immunodetection of this protein on formalin-fixed and paraffin-embedded specimens. They further suggest its routine use in the evaluation of the frequently unpredictable behavior of lung cancer.

The COOH-terminal region of pRb2/p130 binds to histone deacetylase 1 (HDAC1), enhancing transcriptional repression of the E2F-dependent cyclin A promoter.

The tumor suppressor retinoblastoma protein family members pRb, p107, and pRb2/p130 are potent negative transcriptional regulators. The best understood target is the transcription factor E2F, which activates cell cycle-dependent transcription of genes controlling and promoting the cell division cycle (e.g., cyclin A). pRb2/p130 is known to be important in implementing cell cycle exit into G0 due to serum deprivation or various differentiation programs. Several recent studies demonstrated the effect histone acetylases and histone deacetylases (HDACs) have on fine-tuning transcriptional regulation of eucaryotic cells. In this study, we demonstrate that pRb2/p130 binds to HDAC1. This interaction increases the ability of pRb2/p130 to inhibit transcription of the E2F-dependent cyclin A promoter in vivo. We also identify the COOH-terminal 35aa as being necessary for stable interaction between HDAC1 and pRb2/p130.

Cloning of murine CDK9/PITALRE and its tissue-specific expression in development.

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.

A unique domain of pRb2/p130 acts as an inhibitor of Cdk2 kinase activity.

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.

The retinoblastoma gene family pRb/p105, p107, pRb2/p130 and simian virus-40 large T-antigen in human mesotheliomas.

The oncoprotein of simian virus-40, SV40 large T-antigen (Tag), is reported to target and to inactivate growth suppressive proteins such as the retinoblastoma family and p53 (ref. 4, 5), leading to transformation of human cell lines in vitro, tumor production in rodents, and detection of Tag in several human cancers including mesotheliomas. The retinoblastoma family contains three members, pRb, p107 and pRb2/p130 (ref. 9), that are phosphorylated in a cell cycle-dependent manner, have cell growth suppressive properties and bind to specific members of the E2F family and various cyclins. Even though mesotheliomas are among the most aggressive human cancers, alterations of important cell-cycle "controllers," such as the Rb family genes, have never been reported in these tumors. We found the presence of SV40-like sequences in 86% of 35 archival specimens of mesothelioma. We also demonstrated that SV40 Tag, isolated from frozen biopsies of human mesothelioma, binds each of the retinoblastoma family proteins, pRb, p107 and pRb2/p130, in four of four specimens. We propose that the tumorigenic potential of SV40 Tag in some human mesotheliomas may arise from its ability to interact with and thereby inactivate several tumor and/or growth suppressive proteins.

Biological characterization of two novel cathelicidin-derived peptides and identification of structural requirements for their antimicrobial and cell lytic activities.

Cathelicidins are a family of myeloid antimicrobial peptide precursors that have been identified in several mammalian species (Zanetti, M., Gennaro, R., and Romeo, D. (1995) FEBS Lett. 374, 1-5). Two novel bovine congeners have been deduced from cDNA. Their C-terminal sequences of 27 and 28 residues correspond to putative antimicrobial peptides with a cationic N-terminal region predicted to assume an amphipathic alpha-helical conformation followed by a hydrophobic C-terminal tail. Peptides corresponding to these sequences have been chemically synthesized and shown to exert a potent antimicrobial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, and fungi. Both peptides are also cytotoxic to human erythrocytes and neutrophils, although at higher than microbicidal concentrations. The target selectivity has been improved by synthesizing truncated analogues, comprising only the 18 N-terminal residues, which show a great reduction in cytotoxic, but not in antimicrobial activity. The involvement of the C-terminal hydrophobic tail in the cytotoxic activity has been further demonstrated by inducing a major loss of activity in an analogue after replacing highly hydrophobic residues with more hydrophilic ones.

Molecular cloning, expression, and developmental characterization of the murine retinoblastoma-related gene Rb2/p130.

The product of the retinoblastoma-related human gene Rb2/p130 is highly homologous with the product of the retinoblastoma tumor suppressor gene (pRb) and Rb-related p107. this homology is shared mainly in the pocket domain, a region that seems to play a key role in the functions of these proteins. Here we report the molecular cloning and initial characterization of the cDNA encoding the murine homologue of the human Rb2/p130 gene product. The 4.8-kb cDNA encodes a protein of 1125 amino acids that shows 90% indentity to that of the human protein. The Rb2/p130 mRNA is found to be expressed in all of the adult mouse tissues examined, with the highest level being detected in kidney and skeletal muscle. For the protein characterization, we used a polyclonal antibody raised against the COOH terminus of the human Rb2/p130 protein that also recognizes the mouse protein. In developing mouse embryos, the Rb2/p130 protein is expressed as early as day 10 of gestation and reached a peak of expression around day 13 of gestation, implying a developmental regulation of the Rb2/p130 gene in murine ontogeny.

cDNA sequences of three sheep myeloid cathelicidins.

Several myeloid antimicrobial peptide precursors have been shown to consist of a N-terminal proregion similar to a protein named cathelin and a structurally varied C-terminal antimicrobial domain. Proteins with these features have been named cathelicidins. In this paper we report the cDNA sequences of three ovine cathelicidins of 155, 160 and 190 residues, respectively, with cationic C-terminal sequences corresponding to putative antimicrobial domains. These are structurally varied and include a Cys-rich sequence of 12 residues, which is similar to the bovine antimicrobial cyclic dodecapeptide, a novel 29 residue sequence named SMAP-29 with a possible alpha-helical conformation, and a 60 residue sequence named Bac7.5, which appears to be a new member of the Pro- and Arg-rich group of mammalian antimicrobial peptides.