DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

Adult mesenchymal stromal stem cells for therapeutic applications.

Mesenchymal stromal stem cells (MSC) can be found in almost any adult organ. They can be isolated and expanded within several weeks up to hundreds of millions of cells. The cell isolation based on the surface antigen expression may significantly enrich for the desired cell population and reduce the time required for cell expansion. MSC display a unique molecular signature which clearly discriminates them from other stem cell types. MSC can be differentiated into the cells of several lineages. Additionally, the unique biological properties of MSC are mediated by strong immunomodulatory activity and by paracrine mechanisms. Potential therapeutic applications of the cells require clinically compliant protocols for cell isolation and expansion. The therapeutic utility of MSC has been evaluated and found to be useful in several pre-clinical animal models as well as in clinical trials.

Opposite transcriptional effects of cyclic AMP-responsive elements in confluent or p27KIP-overexpressing cells versus serum-starved or growing cells.

The minute virus of mice, an autonomous parvovirus, requires entry of host cells into the S phase of the cell cycle for its DNA to be amplified and its genes expressed. This work focuses on the P4 promoter of this parvovirus, which directs expression of the transcription unit encoding the parvoviral nonstructural polypeptides. These notably include protein NS1, necessary for the S-phase-dependent burst of parvoviral DNA amplification and gene expression. The activity of the P4 promoter is shown to be regulated in a cell cycle-dependent manner. At the G1/S-phase transition, the promoter is activated via a cis-acting DNA element which interacts with phase-specific complexes containing the cellular transcription factor E2F. It is inhibited, on the other hand, in cells arrested in G1 due to contact inhibition. This inhibitory effect is not observed in serum-starved cells. It is mediated in cis by cyclic AMP response elements (CREs). Unlike serum-starved cells, confluent cells accumulate the cyclin-dependent kinase inhibitor p27, suggesting that the switch from CRE-mediated activation to CRE-mediated repression involves the p27 protein. Accordingly, plasmid-driven overexpression of p27 causes down-modulation of promoter P4 in growing cells, depending on the presence of at least two functional CREs. No such effect is observed with two other cyclin-dependent kinase inhibitors, p16 and p21. Given the importance of P4-driven synthesis of protein NS1 in parvoviral DNA amplification and gene expression, the stringent S-phase dependency of promoter P4 is likely a major determinant of the absolute requirement of the minute virus of mice for host cell proliferation.

Expression of E1A in terminally differentiated muscle cells reactivates the cell cycle and suppresses tissue-specific genes by separable mechanisms.

Terminally differentiated cells are characterized by permanent withdrawal from the cell cycle; they do not enter S phase even when stimulated by growth factors or retroviral oncogenes. We have shown, however, that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. In this report, we describe the molecular events triggered by E1A in terminally differentiated skeletal muscle cells. We found that in myotubes infected with the adenovirus mutant dl520, 12S E1A bypasses the early G1 phase and activates the expression of late-G1 genes, such as the cyclin E and cyclin A genes, cdk2, PCNA, and B-myb. Of these, the cyclin E gene and cdk2 were significantly overexpressed in comparison with levels in proliferating, undifferentiated myoblasts. p130 and pRb were phosphorylated before the infected myotubes entered S phase, despite the high expression of the cyclin-dependent kinase inhibitor p21, and E2F was released. Our results suggest that one of the mechanisms that E1A uses to overcome the proliferative block of terminally differentiated cells involves coordinated overexpression of cyclin E and cdk2. Following E1A expression, the myogenic transcription factors MyoD and myogenin and the muscle-specific structural genes encoding muscle creatine kinase and myosin heavy chain were downregulated. The muscle regulatory factors were also silenced in myotubes infected with adenovirus E1A mutants incapable of reactivating the cell cycle in terminally differentiated muscle cells. Thus, the suppression of the differentiation program is not a consequence of cell cycle reactivation in myotubes, and it is induced by an independent mechanism. Our results show that E1A reactivates the cell cycle and suppresses tissue-specific gene expression in terminally differentiated muscle cells, thus causing dedifferentiation.

Cell cycle regulation of the murine cyclin E gene depends on an E2F binding site in the promoter.

Cyclin E controls progression through the G1 phase of the cell cycle in mammalian fibroblasts and potentially in many other cell types. Cyclin E is a rate-limiting activator of cdk2 kinase in late G1. The abundance of cyclin E is controlled by phase-specific fluctuations in the mRNA level; in mammalian fibroblasts, mRNA is not detected under conditions of serum starvation and is accumulated upon serum stimulation, with expression starting in mid-G1. Here, we report the cloning of the murine cyclin E promoter. We isolated a 3.8-kb genomic fragment that contains several transcriptional start sites and confers cell cycle regulation on a luciferase reporter gene. This fragment also supports transcriptional activation by adenovirus E1A, a known upstream regulator of cyclin E gene expression. An E2F binding site which is required for G1-specific activation of the cyclin E promoter in synchronized NIH 3T3 cells was identified in this fragment.

Activation of the E2F transcription factor by cyclin D1 is blocked by p16INK4, the product of the putative tumor suppressor gene MTS1.

The oncoprotein cyclin D1 binds to and activates cyclin-dependent kinase 4 (cdk4), whose activity is inhibited by p16INK4, the product of the putative tumor suppressor gene MTS1. Cyclin D1 controls the timing of S phase onset in mammalian cells. We show that cyclin D1 acts as a positive regulator of the transcription factor E2F. In particular, cyclin D1 overexpression leads to the activation of the dihydrofolate reductase (DHFR) gene promoter. Activation depends on the E2F binding site in the DHFR promoter, known to mediate its activation at the G1/S transition in vivo. Cyclin D1 can also activate the adenovirus E2 promoter via E2F. Both promoters are repressed by p16INK4 and this repression can be released by overexpression of cdk4. The data reported here support a direct role for cyclin D1 and its associated kinase in cell cycle regulation of E2F activity and S phase-specific gene expression. In addition, we show that both E2F sites bind complexes containing the retinoblastoma protein (pRB) and that in RB-deficient cell lines overexpression of cyclin D1 fails to activate E2F-dependent transcription, indicating that pRB may be involved in promoter activation.

S-phase induction by adenovirus E1A requires activation of cdc25a tyrosine phosphatase.

Adenovirus E1A proteins can induce quiescent cells to enter S-phase and also affect the expression of cellular genes including various cell cycle regulators. Here we show that human cdc25A, a tyrosine phosphatase involved in regulation of the G1/S-phase transition of the cell cycle, is a target of the adenovirus E1A protein in virus-infected human fibroblasts. Expression of E1A in quiescent fibroblasts leads to a rapid increase in cdc25A phosphatase activity and also increases both cdc25A and cyclin E gene expression. Inhibition of cdc25A function by antibody injection prevents virus-induced entry into S-phase. These results indicate that induction of high levels of cdc25A and its potential positive regulator cyclin E mediates the ability of E1A to induce S-phase in the presence of antiproliferative signals.

The role of p53 in coordinated regulation of cyclin D1 and p21 gene expression by the adenovirus E1A and E1B oncogenes.

Expression of the cyclin D1 gene is induced when quiescent fibroblasts are stimulated to reenter the cell cycle by addition of growth factors. Moderate ectopic expression of cyclin D1 in early G1 facilitates progression through G1. When transiently overexpressed at the G1/S boundary, cyclin D1 prevents S phase entry, suggesting a dual role for this protein in cellular growth control. It was shown that the retinoblastoma protein (pRB) can activate cyclin D1 gene expression; furthermore, there is evidence that expression of the cyclin D1 gene is down-regulated by the SV40 large T and adenovirus E1A genes, both of which were shown to target pRB. We now report that in diploid human fibroblasts functional inactivation of pRB by adenovirus E1A is not sufficient for efficient repression of cyclin D1 gene expression, since the E1B gene product, in addition to E1A, is required for repression of the cyclin D1 gene. Since E1B was shown to target p53, we investigated the role of p53 for expression of the cyclin D1 gene. In a cell line with temperature-sensitive p53, cyclin D1 is moderately expressed at the restrictive temperature. Induction of p53 function by temperature shift leads to an increase of cyclin D1 mRNA and protein, parallel to the activation of p21WAF-1/CIP1 gene expression in this system. When the capability of adenovirus gene products to affect expression of either gene was analysed, we found that infection of Ad5 drastically reduced cyclin D1 and p21WAF-1/CIP1 gene expression in cells where p53 function is limiting. Under these conditions E1A and E1B cooperate to reduce the cyclin D1 level, while p21WAF-1/CIP1 expression was found insensitive to E1A expression. In cells containing elevated p53 function, modulation of gene expression by E1B was severely compromised; under these conditions, expression of E1A reduced expression of cyclin D1 without affecting p21WAF-1/CIP1. The data suggest that E1A and E1B cooperate to inhibit expression of cyclin D1 and identify the cyclin D1 gene as a new downstream target for p53.

p53-independent growth regulation of cervical cancer cells by the papillomavirus E6 oncogene.

Growth of cervical carcinoma cells depends on continuous expression of high risk type human papillomavirus oncogenes E6 and E7. E6 destabilizes p53, a tumor-suppressive transcription factor, which activates expression of the inhibitor of cell cycle progression p21 and other genes. E6-mediated p53 degradation can therefore result in cell cycle deregulation. It has, however, not yet been determined whether p53 inactivation is sufficient to provoke cell cycle progression in established cervical carcinoma cells. Moreover, it has not yet been clarified whether E6 confers additional p53-independent growth stimuli in cancer cells. To address these questions, we analysed p53 functions in SW 756 cervical cancer cells in which the expression of endogenous HPV 18 E6-E7 genes can be downregulated by dexamethasone. This results in significantly increased p53 levels and subsequent cell cycle arrest in the Gz phase. Surprisingly, p53 activities were suppressed rather than enhanced in these cervical cancer cells. However, if high risk papillomavirus type 16 E6 genes, including a mutant which does not degrade p53, were expressed in dexamethasone-treated SW 756 cells with suppressed endogenous HPV type 18 E6-E7 expression, the cells reentered the cell cycle even in the absence of a cooperating viral E7 gene. In contrast, the non oncogenic papillomavirus type 6 E6 gene did not release the cells from growth arrest under these conditions. These data indicate that suppression of p53 functions is not sufficient to provoke cell cycle progression in E6-E7-depleted cervical cancer cells and point to a p53-independent mitotic activity to oncogenic papillomavirus type E6 genes in cervical carcinoma cells.

Functional p53 protein in human papillomavirus-positive cancer cells.

There is accumulating evidence that the p53 protein contributes to tumor suppression by stimulating the transcription of specific cellular genes, such as the cell cycle control gene WAF1/ClP1. p53-mediated transcriptional activation is inhibited in cotransfection assays by overexpressed E6 protein from cancer-associated human papillomavirus (HPV) types, pointing at a possible molecular mechanism by which these viruses contribute to malignant cell transformation. Here we analysed the transcriptional transactivation function of endogenous p53 protein in a series of cervical cancer cell lines, which express the E6 gene from integrated viral sequences. Transient and stable transfection analyses employing p53-responsive reporter constructs indicated that HPV-positive cervical cancer cells contained transactivating p53 protein. Treatment of HPV-positive cells with genotoxic agents, such as mitomycin C, cisplatin, or u.v. irradiation, resulted in an increase of nuclear p53 protein levels and enhanced binding of p53 to a p53-recognition site. These effects were accompanied by an increase of WAF1/ClP1 mRNA levels. In several HPV-positive cell lines, these molecular events were linked to a cell cycle arrest in G1. In contrast, cancer cells containing mutant p53 genes did not contain transactivating endogenous p53 protein and lacked the p53-mediated response to DNA damaging agents. These results indicate that the tumorigenic phenotype of HPV-positive cancer cell lines does not necessarily correlate with a lack of basal or DNA damage induced p53 activities and that therefore the presence of high risk HPV sequences is not functionally equivalent to the loss of p53 function through somatic mutations of the p53 gene.

The human papillomavirus type 16 E7 protein is associated with the nucleolus in mammalian and yeast cells.

In this study we show, by immunofluorescence and electron microscopy immuno-gold labelling, that the major transforming protein of Human Papillomavirus type 16 E7 is associated with the nucleolus of cells derived from the HPV16-positive cervical carcinoma line CaSki. The E7 nucleolar staining appeared to be cell cycle dependent, being considerably reduced in the G2 phase. The total level of the protein in the cell, however, remained constant during all phases. We also show that the cellular protein Rb1, which is targeted by E7, is localised in the nucleus and nucleolus in CaSki cells. Thus, it is possible that the presence of E7 in the nucleolus correlates with a hypothetical function(s) of Rb1 in this particular intranuclear compartment. The nucleolar localisation of HPV16 E7 protein was also observed in the fission yeast Schizosaccharomyces pombe, suggesting that a targeting mechanism of HPV16 E7 protein into the nucleolus is common to both mammalian and yeast systems. Nucleolar localisation of HPV16 E7 protein may be independent from Rb1 since no Rb1 related proteins have been identified in fission yeast.

Activated fos oncogene in rat embryo fibroblasts transformed by ras and myc oncogenes.

Rat embryo fibroblasts (REF) were transformed by simultaneous gene transfer of the complementary oncogenes ras and myc using the calcium phosphate coprecipitation method. Cell lines derived from transformation foci expressed in addition to ras and myc cellular oncogene fos while normal REF did not express ras, myc and fos according to the hybridization methods used. The transformed cell lines produced colonies in soft agar and tumors in newborn syngeneic rats. From one tumor a cell line was established which was characterized by a high level of fos gene expression.

Interphase chromosome locus displacement induced by low-doses of radiation.

The most important stage in the making of mutations is a reparation of different DNA damage, including the more deleterious double-strand DNA breaks (DSB). The first stage of adaptive response--fundamental antimutagenic cell reaction, purposeful to reparation for induced DSB repair--is investigated in present work. Non-radioactive in situ hybridization of biotin-labeled DNA probe was used to mark chromosome 1 pericentromeric regions (PR) in G0 human lymphocytes. It was shown that under 3-10 cGy (X-radiation, 160 kV) PR become displaced from a nucleus periphery to inner territory of a nucleus. The moving process realizes during several hours after an irradiation. As far as some non-specific gene repressors are co-localized with chromosome centromeric regions it is possible hypothesizes that the displacement cause changing expression of some genes. It is possible to propose that an absence of radiation induced chromosome locus displacement may be one of causes DSB repair disturbance. This hypothesis was tested by the model. It is assumed that one consequence of the underlying defect may be inappropriate involvement of cell's recombination machinery in the repair of DSB. We studied lymphocytes of patients with hereditary BRCA2 mutation. It is thought that this gene takes part in DSB repair. The significant differences of the PR moving between control samples and the cases were revealed under 10 cGy. Similar results were observed on lymphocytes of patients with Fanconi syndrome. Thus, abnormal moving of interphase nucleus chromosomes conditioned by low-dose irradiation may suggest on imperfect machinery of DSB repair, i.e. genetic risk. We realize that further investigations are needed for definitive conclusion.

Telomere DNA sequences and the concept of ontogenetic reserve cells.

Dynamics of telomere sequences are considered in normal and immortalized cells. Immortalized cells are suggested to be derived mainly from a special subpopulation of ontogenetic reserve cells. Their epigenetic program consists of autoregeneration during external stimulus for genome reorganization and corresponding appearance of genetic variants of cells. It is suggested that cells surviving the crisis stage contain a special signal sequence integrated into telomere DNA. Its elimination during shortening of DNA telomere sequences in dividing ontogenetic reserve cells is a signal for the cooperative transition of chromatin into a new steady state that corresponds to the epigenotype of immortalized cells. Localization of telomere DNA sequences in intrachromosomal "hot spots" reflects phylogenetic rearrangement of the genome.

The effect of heparin on the porcine lymphocyte chromatin--I. The comparative study of DNA in different chromatin fractions.

1. Porcine lymphocyte chromatin in the solution of 0.15 M NaCl + 0.01 M Tris, pH 7 treated with heparin liberated 30% protein and 7.5% DNA to the supernatant. 2. DNA from the supernatant and the pellet fractions as well as from control chromatin were isolated in identical conditions. 3. No significant changes were observed in spectral properties and melting points in SSC of comparable DNA specimens. 4. It was noted, however, that DNA of the supernatant is subject to denaturation in the process of isolation, which, apart from the difference in protein composition of the supernatant and the pellet fractions, suggests different chromatin structure of these fractions.

Chromatin structure in Down's syndrome.

In the chromatin of patients with Down's syndrome, changes are shown to occur in a short-term lymphocyte culture of the human peripheral blood. Some of them are induced by the patient's blood serum and are reversible when this is replaced by normal serum. A 100-fold dilution of the blood serum taken in subjects with Down's syndrome does not produce any changes in the structure of the lymphocyte chromatin of the patients. A similar procedure with the blood serum of healthy donors resulted in a drastic activation of their lymphocyte chromatin. These experiments, and investigations on the effect produced by the blood serum on the model desoxyribonucleoprotein systems, support the suggestion that the changed state of the chromatin in subjects with Down's syndrome is caused by a complex set of components contained in the blood serum, whose degree of dissociation deviates from the normal.

Down-regulation of cyclin A gene expression upon genotoxic stress correlates with reduced binding of free E2F to the promoter.

Treatment of mammalian cells by DNA-damaging agents leads to various cellular responses. At sufficiently high dosage, cisplatin blocks cell proliferation and finally kills cells; this effect is the basis for its widespread use as an anticancer drug. Cisplatin-treated cells arrest in the G1 phase of the cell cycle, most likely due to a signal generated by the stabilization of p53 and the subsequent induction of p21WAF-1/Cip1. We show here that cisplatin-treated mammalian cells accumulate normal levels of cyclin D1 and cyclin E but fail to produce cyclin A. The block to cyclin A gene expression occurs at the level of transcription and is mediated by an E2F binding site in the cyclin A promoter. It is shown here that, upon cisplatin treatment, transcriptionally active free E2F becomes limiting, coincident with the accumulation of hypophosphorylated species of the retinoblastoma protein family. Immunoprecipitation experiments suggest that the loss of free E2F results, at least in part, from the sequestration of E2F-4/DP-1 heterodimers by p107. A role for the kinase inhibitor p21WAF-1/Cip1 in repression of the cyclin A promoter is supported by our finding that ectopic expression of p21WAF-1/Cip1 is sufficient to inhibit transcription from the cyclin A gene, dependent on the E2F site. The data establish the E2F site in the human cyclin A promoter as a key target for the signaling pathway leading to G1 arrest in response to DNA damage by cisplatin and potentially other genotoxic agents.

Transformation of rat-embryo immortalized fibroblasts by the E6-E7 region of human papillomavirus type 18.

Plasmids containing the E6 and E7 open reading frames of human papillomavirus type 18 transformed rat-embryo fibroblasts when expressed under the cytomegalovirus promoter. The fibroblasts had been previously immortalized with the large T-antigen gene of the polyomavirus to produce rat embryo fibroblast (large T-antigen) [REF(LT)] cells. REF(LT) cells were transformed by the E6 and E7 sequences to anchorage independence and tumourigenicity, but there were no significant morphological alterations. Transformation by these sequences of REF(LT) cells differed from that achieved by pEJras, in which case significant morphological changes and tumourigenicity in nude mice did occur.

Interactions between steroid hormones and viral oncogenes in the pathogenesis of cervical cancer.

Steroid hormones are frequently prescribed as anticontraceptive drugs to young women. Persistent papillomavirus infections particularly with high risk virus types are very common in younger women and were shown to be the strongest risk factor for the later development of cervical cancer. Steroid hormones interfere with persistent papillomavirus infections on various levels. They enhance the expression level of two viral genes, E6 and E7, which are required for the oncogenic activities of high risk papillomaviruses. In addition, they interfere with cellular gene functions involved in cell cycle regulation and programmed cell death, for example through inhibition of p53-mediated transcriptional transactivation of genes involved in cell cycle arrest and apoptosis. Furthermore, steroids inhibit the immunologically mediated resolution of minor HPV-induced cervical lesions, particularly through inhibition of major histocompatibility class I and class II antigen expression. These observations point to potent cocarcinogenic effects of steroid hormones in persistently papillomavirus infected individuals by enhancing the transforming activities of viral oncogenes and interfering with the efficient resolution of virus infected lesions. The clinical significance of these experimental observations requires careful analysis in prospective trials.

Adenovirus E1A activates cyclin A gene transcription in the absence of growth factors through interaction with p107.

Using the infection of quiescent human fibroblasts with adenovirus type 5 and various deletion mutants, we show that E1A can stimulate transcription of the cyclin A gene in the absence of exogenous growth factors. Required for this activity is conserved region 2 (CR2), while both the N-terminal part of E1A and CR1 are dispensable. This indicates that activation of cyclin A gene expression requires the binding of E1A to p107, while binding to either pRB or p300 is not involved in transcriptional activation. We demonstrate that p107 represses the cyclin A promoter through its cell cycle-regulatory E2F binding site and that 12S E1A can activate the cyclin A promoter, essentially by counteracting its repression by p107. Since Cr2 is required for cell transformation, transcriptional activation of the cyclin A gene by E1A appears to be important for its capacity to override control of cellular growth.