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.

Critical role played by cyclin D3 in the MyoD-mediated arrest of cell cycle during myoblast differentiation.

During the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and with the requirement of the p300 transcriptional coactivator. In differentiated myocytes, cyclin D3 also becomes stabilized and is found nearly totally complexed with unphosphorylated pRb. The detection of complexes containing cyclin D3, cdk4, p21, and PCNA suggests that cdk4, along with PCNA, may get sequestered into high-order structures held together by pRb and cyclin D3. Cyclin D3 up-regulation and stabilization is inhibited by adenovirus E1A, and this correlates with the ability of E1A to promote pRb phosphorylation; conversely, the overexpression of cyclin D3 in differentiated myotubes counteracts the E1A-mediated reactivation of DNA synthesis. These results indicate that cyclin D3 critically contributes to the irreversible exit of differentiating myoblasts from the cell cycle.

Lack of correlation between N-myc and MAX expression in neuroblastoma tumors and in cell lines: implication for N-myc-MAX complex formation.

Detectable levels of MAX messenger RNA were found in a set of human neuroblastoma tumors and established cell lines. MAX mRNA levels were independent of tumor stage and N-myc genomic amplification. By contrast, N-myc mRNA transcripts were detectable only in tumors with amplification of N-myc gene and in cell lines. Analysis by reverse transcriptase polymerase chain reaction and hybridization to specific oligodeoxynucleotide probes revealed approximately equal amounts of two MAX transcripts in all cases analyzed. Immunoprecipitations with a specific antibody to MAX detected two proteins of M(r) 21,000 and 22,000 in approximately equal amounts in all neuroblastoma lines regardless of N-myc amplification and/or expression. On the other hand, protein binding to the myc DNA consensus sequence correlated with N-myc expression in neuroblastoma cells. Thus, N-myc expression might be a limiting factor in the formation of the N-myc-MAX heterodimer in neuroblastomas.

Defective human retinoblastoma protein identified by lack of interaction with the E1A oncoprotein.

Inactivating mutations of the retinoblastoma susceptibility gene (Rb) are involved in the pathogenesis of hereditary and sporadic retinoblastoma. Alterations in the Rb gene have also been found in several other human tumors occurring with epidemiological incidence higher than that of retinoblastoma. Four human malignant glioma cell lines were examined for abnormalities in the retinoblastoma gene product (pRb), using a procedure based on the interaction of pRb with an in vitro-translated adenovirus E1A oncoprotein. In the CRS-A2 cell line, derived from a glioblastoma multiforme, pRb did not bind with the in vitro-translated E1A protein. Restriction analysis of the CRS-A2 Rb gene and Rb mRNA expression provided patterns that could not be distinguished from the other glioma cell lines. Further investigation revealed the presence of a truncated pRb in the CRS-A2 cell line, due to a nucleotide insertion in the coding sequence at position 2550. In addition, this truncated Rb protein was undetectable in phosphorylated form. The binding assay with the in vitro-translated E1A was also used to study other cell lines with known mutations in the Rb gene. This method, which evaluates the interaction between in vitro-translated E1A and the pRb, is proposed as a rapid screening for detecting functional alterations in the retinoblastoma protein.

The retinoblastoma gene product in acute myeloid leukemia: a possible involvement in promyelocytic leukemia.

The retinoblastoma susceptibility gene in leukemia and lymphoma has been investigated using different approaches involving either gene or protein analysis. In this study, a novel method, which evaluates the functional status of the retinoblastoma gene product by a binding assay to an in vitro-translated viral oncoprotein, has been applied to leukemic cells from acute myeloid leukemia patients. One hundred twenty-two cases were considered, and 42 of them were also analyzed by Western blot. Results obtained with the two methods were comparable, with the exception of few cases, where the retinoblastoma protein appeared detectable but unable to bind to the viral oncoprotein. The retinoblastoma protein has been found defective mostly in the M3 promyelocytic subtype.

Regulation of MyoD gene transcription and protein function by the transforming domains of the adenovirus E1A oncoprotein.

It has been demonstrated that the adenovirus E1A gene products inhibit myogenic differentiation in the mouse C2 muscle cell line. During myogenic differentiation, cell growth and tissue-specific gene expression are mutually exclusive. Since E1A exerts multiple effects on different cellular pathways through alteration of cell growth control and transcriptional regulation, we investigated in more detail the molecular mechanisms underlying the inhibitory effect of E1A on myogenic differentiation. To this end, we used mutant derivatives of E1A that lack the 'conserved domain' sequences to which the functional domains of E1A have been mapped, and we observed the effect of constitutive expression of these E1A mutants on myogenesis in the murine C2 muscle cell line. Our results demonstrate that E1A interferes with myogenesis through at least two mechanisms: (i) the inhibition of MyoD expression; (ii) the repression of MyoD-dependent transcriptional activation. In addition, we demonstrate also that the repression of MyoD transcription depends upon sequences located in the N-terminus of E1A and correlates well with the site of E1A/p300 association. Further, the inhibition of transcriptional activation by MyoD depends both on conserved region 1 and on conserved region 2, the two transforming domains of E1A. We demonstrate also that a similar inhibitory effect on the MyoD transactivating function is provided by the polyomavirus and SV40 large T oncoproteins.

MyoD induces retinoblastoma gene expression during myogenic differentiation.

In skeletal muscle cells permanent withdrawal from the cell cycle is a prerequisite for terminal differentiation. The muscle-specific transcription factor MyoD can activate downstream muscle structural genes and myogenic conversion in many different cell types. It has been demonstrated that the product of the retinoblastoma susceptibility gene, with its growth-suppressive activity, is involved in the myogenic function of MyoD (Caruso et al., 1993; Gu et al., 1993). The present study characterises the modulation of retinoblastoma (Rb1) mRNA levels during myogenic differentiation of the murine C2 cell line and provides evidence that the muscle-specific regulatory factor MyoD enhances Rb1 gene transcription. We demonstrate that MyoD mediates the transactivation of a CAT construct whose expression is driven by the human Rb1 gene promoter, and that this is not a consequence of direct binding of MyoD to an E-box DNA sequence motif present in the Rb1 promoter sequences. In addition we have tested the capability of several MyoD mutant proteins of inducing the Rb1 promoter CAT construct. Our results indicate that the MyoD function required for induction of Rb1 promoter activity is distinct from its myogenic function.

Myc down-regulation induces apoptosis in M14 melanoma cells by increasing p27(kip1) levels.

In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the melanoma pathogenesis. We have previously demonstrated that treatment of melanoma cells with c-myc antisense oligodeoxynucleotides can inhibit cell proliferation and activate apoptosis. To gain insight into the mechanisms activated by Myc down-regulation, we have now developed an experimental model that allows modulating Myc protein expression in melanoma cells. This was achieved by originating stable melanoma cell clones expressing ecdysone-inducible c-myc antisense RNA. We show that the induction of c-myc antisense RNA in M14 melanoma cells leads to an inhibition of cell proliferation characterized by accumulation of cells in the G(1) phase of the cell cycle (up to 80%) and activation of apoptosis (50%). These data are associated with an increase of p27(kip1) levels and a significant reduction of the cdk2-associated kinase activity. In addition, we show that an ectopic overexpression of p27(kip1) in this experimental model can enhance the apoptotic rate. Our results indicate that down-regulation of Myc protein induces a G(1) arrest and activates apoptosis by increasing p27(kip1) content in melanoma cells, that are known to be defective for the p16-cyclinD/cdk4-pRb G(1) checkpoint. This is particularly relevant for identifying new therapeutic strategies based on the re-establishment of the apoptotic pathways in cancer cells.

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.

MYCN gene expression is required for the onset of the differentiation programme in neuroblastoma cells.

Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma differentiation to induce apoptosis in cells that are not committed to differentiate.

Sites hypersensitive to, and protected from, nuclease digestion in the regulatory region of wild-type and mutant polyoma chromatin.

It has been shown that the untranscribed regulatory region of polyoma virus (Py) is hypersensitive (Hs) to DNase I treatment, and that this hypersensitivity is located in two areas which correspond to the A and B domains of the enhancer. We mapped the DNase I hypersensitive sites in the Py regulatory region of wild-type (PyA2) and of mutants, selected in neuroblastoma cells (PyNB), which are characterized by an extensive duplication involving the A domain, with or without deletion of the B domain. The experiments were performed in both a permissive host (3T6 mouse fibroblasts) and in a restrictive host (41A3 mouse neuroblasts). No significant differences were observed between the two hosts. Our results show that four sites, in addition to the ones already described, can be identified in the wild-type A2 strain. These newly identified sites coincide with the domains of the enhancer region as they have recently been established. In PyNB mutants duplications and deletions are generally correlated to the gain or loss of the corresponding hypersensitive sites. However, a new site is formed in one of the duplicated sequences, even if no corresponding hypersensitive site is present in the other identical sequence. A region protected from DNase I digestion occurs in the PyNB mutants which corresponds to the junction of the duplication which is absent in the wild-type strain. In this region, as a consequence of the rearrangement, a GGCGGG motif which is very similar to the one (GGGCGG) present at the binding sites of the cellular regulatory protein SP1, is found.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse genes coding for "zinc-finger"-containing proteins: characterization and expression in differentiated cells.

By using the zinc-finger region of human cHF.10 cDNA as a probe at low-stringency hybridization conditions, several individual phages from a mouse skeletal muscle cDNA library have been isolated. The amino acid sequences of the "zinc-finger" domains derived from the DNA sequences of three cDNA clones are shown. The expression of the corresponding mRNAs in three cell lines (NIH 3T3, F9 teratocarcinoma, and C2 myoblast cells) at different stages of differentiation and in eight adult mouse tissues has been analyzed. The transcription of these genes is induced during the in vitro differentiation of the cell lines tested. These three genes are widely and evenly expressed in adult mouse tissues, with the remarkable exception of one that is expressed predominantly in the testis.

Characterization of two novel YY1 binding sites in the polyomavirus late promoter.

NF-D is a ubiquitous nuclear factor that has been shown to bind specifically to a DNA element in the polyomavirus regulatory region. In this report, we demonstrate that NF-D is either identical or very similar to a transcription factor that has been variously named YY1, delta, NF-E1, UCRBP, or CF1. Moreover, we show the presence in the polyomavirus genome of a second DNA motif, located 40 bp from the first, which binds YY1/NF-D with high affinity. Both sites lie downstream of the major late transcription initiation sites. By site-directed mutagenesis, we demonstrate that both elements contribute positively to the activity of the late promoter, probably by a cooperative mechanism. We also demonstrate that the requirement of the YY1/NF-D function for late promoter activity varies with the cell line.

Poly(A)-containing RNA in neuroblastoma: immature and differentiated cells in culture.

We have analysed the poly(A)-containing RNA from neuroblastoma cells at two different developmental states: either as round, immature neuroblasts grown in suspension, or as differentiated cells exhibiting the morphological properties of mature neurons, when attached to a culture dish. Suspension-grown and monolayer cells were pulse-labelled with tritiated uridine. The profile of cytoplasmic poly(A)-containing RNA from suspension cells is highly heterogeneous with peaks ranging from 16-30 S. The profile obtained from differentiated cells appears somewhat distinct from the previous one. This is evidenced by a relative decrease in the 26-S peak and a virtual disappearance of the 16-S component. In order to compare the 'steady-state' patterns of poly(A)-containing RNA in these two developmental stages, polysomal RNA was prepared from unlabelled cells. Following sucrose gradient sedimentation, each fraction was hybridized to [3H]poly(U). Examination of the two RNA hybridization profiles reveals striking similarities suggesting that 'steady-state' messenger populations include, on the average, the same subspecies. The 16-S fraction, which was not observed after the pulse-labelling of the monolayer culture, is detected here by hybridization to [3H]poly(U) when using polysomal poly(A)-containing RNA from monolayer cells as substrate. These results suggest that terminal differentiation of neuroblastoma cells is not accompanied by major alterations of the transcription program and is paralleled by a marked stabilization of the 16-S species.

Expression in male and genomic organization of the gene(s) coding for a major protein secreted by the rat seminal vesicle epithelium.

Double strand cDNA copies of lls poly(A)+mRNA purified from adult rat seminal vesicles (RSV), have been cloned in E.coli C600 using the Pst I site of pBR322. Filter hybridization, nucleotide sequence analysis and positive hybridization translation were used to demonstrate that one of the recombinant plasmids obtained (pRSV25) contained a 260 bp long insert coding for a significant part of the precursor to the protein IV present in the RSV secretion. By using labelled pRSV25 DNA we have found that high levels of RSV IV mRNA were present only in the rat seminal vesicle epithelium. The amounts of RSV IV mRNA present in other tissues of the same organism were below the levels detectable by the methods used. In addition, other data reported here indicate that the RSV IV gene(s) is present in both sexes, probably with a different organization.