Enhanced activity of cloned hamster TERT gene promoter in transformed cells.

In 7,12-dimethylbenz[a]anthracene-treated hamster pouch epithelial cells, telomerase activity increased within 1 week of treatment and reached a 6-7-fold increase within 3 weeks. To investigate this phenomenon, we have cloned and sequenced the hamster telomerase catalytic subunit (hamTERT) promoter. Transient transfection with different genomic segments upstream of the ATG translation initiation codon linked to the luciferase reporter gene mapped the core promoter within a 250 bp region. Three major transcription initiation sites and several minor sites were found between -42 and -140 bp relative to the ATG site. Like the human and murine TERT promoters, the hamTERT promoter lacks TATA and CAT boxes and all three promoters share similar regulatory factor binding sites. DNase I footprint analysis revealed six protected regions which contain sequences homologous with known transcription factor binding sites. Three protein binding regions (I, II, and III) were essential for the promoter activity. Regions I and III bound to Sp1 and Sp3 transcriptional factors, whereas region II bound to an unknown factor. Transient transfection of a promoter-luciferase plasmid into Drosophila SL2 cells showed that Sp1 and Sp3 regulated the hamster TERT promoter in a concentration-dependent and synergistic manner. Telomerase activity showed a 2-4-fold and 8-10-fold increase in immortalized cells and tumor cells, respectively, but hamTERT expression was only increased 1.7-fold and 2.4-fold, respectively, in the same cells.

The temperature sensitive mutant p53-143ala extends in vitro life span, promotes errors in DNA replication and impairs DNA repair in normal human oral keratinocytes.

Many human cancers contain a hemizygous point missense mutation in p53, allowing expression of both wild-type and mutant p53. To understand the relationship between wild-type and mutant p53 in cells, we investigated the influence of a naturally occurring temperature-sensitive mutant p53 (valine to alanine substitution at codon 143: mp53-143ala) on the life span of normal human oral keratinocytes (NHOK) and the expression of wild-type p53. We also investigated the effect of the mutant p53 on the genetic stability of NHOK. The mp53-143ala extended the in vitro life span of NHOK by four-fold, but failed to overcome the M2 crisis stage for immortalization. The mp53-143ala notably suppressed wild-type p53 in NHOK at post-transcriptional levels. Moreover, the mp53-143ala notably increased both spontaneous and genotoxic agent-induced mutation frequency of a shuttle vector in NHOK. These data indicate that mutant p53 induces genetic instability by, in part, inhibiting the expression of wild-type p53 through a dominant negative role in cells expressing both mutant and wild-type p53.

In vitro replication and differentiation of normal human oral keratinocytes.

The replication kinetics and cytological changes of normal human oral keratinocytes (NHOK) isolated from the basal surface of oral epithelial sheet and cultured as dispersed cells in low (0.15 mM) Ca(2+) medium without serum were analyzed. Replicating NHOK were quantitated by cell count and identified by [(3)H]thymidine uptake. Cell morphology was analyzed by phase contrast and transmission electron microscopy, and by cytochemical staining for endogenous beta-galactosidase (beta-gal) activity, involucrin, and cytokeratin types 1 and 10 (K1/K10). Primary NHOK obtained from 15 different donors whose ages ranged from 21 to 62 years consistently showed three distinct phases of replication, i.e., exponential, senescing, and senescent, which were independent of the donors' age. Initially, the cells replicated exponentially for a period of 20 days with a doubling time of 26.6 +/- 3.5 h. They then gradually entered replication arrest over a period of 18 days. The cells underwent a maximum of 22.1 +/- 2.8 population doublings. The onset of gradual replication arrest coincided with an increase in the fraction of cells, which stopped DNA synthesis within a maximum of 48 h and which stained for beta-gal. The fraction of terminally differentiated cells stained for K1/K10 did not increase until nearly all the cells had stopped replicating (senescent phase) and maximal beta-gal staining had been reached. Subsequently, the percentage of beta-gal stained cells actually decreased while the percentage of those stained for K1/K10 increased to a maximum of 80-90% within 2-3 weeks. Exposure of exponentially replicating NHOK to 5-aza-2'-deoxycytidine (5-aza CdR) inhibited DNA replication within 18-48 h and induced terminal differentiation 6 days later. In contrast, exposure of these cells to 1.5 mM Ca(2+) induced expression of involucrin and K1/K10 within 48 h without inhibiting DNA synthesis. Thus, replication arrest preceded differentiation in NHOK serially subcultured in vitro; however, differentiation could be induced without replication arrest.

The E7 oncoprotein of human papillomavirus type 16 interacts with F-actin in vitro and in vivo.

We report here that E7 oncoprotein of human papillomavirus type 16 (HPV-16) forms a complex in vivo and in vitro with actin, one of the components of the cellular cytoskeleton. The in vivo interaction was detected by immunofluorescent staining and confocal microscopic examination of normal human oral keratinocytes (NHOK) and CV-1 cells after transient expression of E7 employing the vaccinia virus-T7 RNA polymerase system and by coimmunoprecipitation from an immortalized, nontumorigenic cell line obtained after transfecting NHOK with the cloned HPV-16 DNA genome. The in vitro interaction was detected by cosedimentation of bacterially expressed E7 phosphorylated with rabbit reticulocyte lysate or purified casein kinase II (CKII) prior to incubation with F-actin. This interaction was inhibited if E7 phosphorylation by the rabbit reticulocyte lysate was prevented with heparin, a CKII inhibitor, or if the amino acids Ser-31 and Ser-32 in E7, which are phosphorylated by CKII, were replaced with amino acids that cannot be phosphorylated. Interestingly, a decrease in the amount of polymerized actin occurred in cells expressing E7.

Differential gene expression in neoplastic and human papillomavirus-immortalized oral keratinocytes.

We have previously demonstrated that normal human oral keratinocytes immortalized by transfection with human papillomavirus type-16 Dna became tumorigenic after exposure to a chemical carcinogen. In an effort to detect differentially regulated genes associated with this transition from the immortal to the malignant phenotype, we employed representational differences analysis (a PCR-coupled subtractive hybridization technique). After analysing 50 colonies, 12 putative messages were identified. Northern analysis comparison using the identified cDNAs as probes was made between normal human oral keratinocyte, papillomavirus-immortalized human oral keratinocytes (HOK-16B), a neoplastic cell line derived from HOK-16B (HOK-16B-BaP-T) and the human oral cancer cell lines Hep-2, SCC-9 and Tu-177. We found that mRNAs encoding for cyclophilin A, c-myc binding protein 1, the heat shock protein 90alpha and one unknown transcript were up-regulated in the oral cancer cell lines analysed as well as in HOK-16B cells. We also detected a downregulation of the mRNAs encoding the skin-derived antileukoproteinase SKALP/elafin, the translationally regulated p23 protein and one unknown transcript. Whether these messages are associated to the neoplastic conversion of human keratinocytes remains to be determined.

Ethanol upregulates the expression of p21 WAF1/CIP1 and prolongs G1 transition via a p53-independent pathway in human epithelial cells.

The control of cell cycle progression is necessary for accuracy in the replication of DNA and the distribution of genetic information to daughter cells. Disturbances in progression of the cell cycle may result in the loss of genomic integrity, a 'hallmark' of cancer cells. Extensive consumption of alcoholic beverages is a risk factor associated with the development of various human epidermoid cancer including oral and pharyngeal squamous cell carcinomas. However, effects of ethanol on cell cycle progression and on the expression of genes associated with the cell cycle have not been studied. We report here that exposure of human epithelial cells to ethanol, at concentration (100-200 mM) that do not cause cell death, (a) does not affect or only reduces slightly the cellular level of p53 protein, (b) upregulates the transcription of the WAF1/CIP1 gene, (c) inhibits the Cdk2 activity, and (d) reduces the rate of cellular proliferation by inducing a delay in G1 phase transition. The results also indicate that, at these non-cytotoxic concentrations, ethanol exhibits its effects through a p53-independent mechanism.

HPV-16 oncogenes E6 and E7 are mutagenic in normal human oral keratinocytes.

The mutation frequency of pS189 shuttle vector plasmids is higher in human oral keratinocytes (NHOK) immortalized with cloned human papillomavirus-16 (HPV-16) genome than in primary normal NHOK (NHOK). To determine whether oncoproteins E6 and E7 of HPV-16 are responsible for the higher mutation frequency of the plasmids, we measured the mutation frequency in NHOK and in NHOK expressing the HPV-16 oncogenes (E6, E7, or E6 plus E7). We also measured the mutation frequency in NHOK expressing the E6 or E7 proteins of the non-oncogenic HPV-6b. The mutation frequency, either background or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced, in NHOK expressing the HPV-16 oncoproteins (E6, E7, or E6 plus E7) was significantly higher than in NHOK. The HPV-16 oncogenes did not alter the nature of the MNNG-induced mutations (G:C-->A:T), but increased the frequency of deletions and insertions with or without MNNG. The background or MNNG-induced mutation frequency in NHOK expressing the HPV-6b E6 or E7 proteins was the same as in NHOK. NHOK and NHOK expressing HPV6b-E6 or E7 were able to arrest the cell cycle and enhance cellular p53, p21(WAF1/CIP1), and Gadd45 levels when exposed to MNNG, whereas NHOK expressing the HPV-16 E6 oncogene did not demonstrate. NHOK expressing HPV-16 E7 were able to enhance cellular p53, p21(WAF1/CIP1), and Gadd45 levels, but failed to arrest cell cycle progression when exposed to MNNG. These data indicate that HPV-16 E6 and E7 oncogenes are mutagenic in human oral keratinocytes and enhance the mutagenic effect of MNNG. However, the E6 and E7 proteins of the 'low risk' HPV-6b did not demonstrate such an ability.

Distribution of alternatively spliced chicken c-myb exon 9A among hematopoietic tissues.

The role of the c-myb proto-oncogene in cellular differentiation may be regulated in part by alternative splicing of its mRNAs. Previously, two forms of alternative splicing of the chicken c-myb gene between exons 9 and 10 were described: one form utilizes the entire 360 base pair (bp) exon 9A while a second form utilizes exon 9A' which consists of the 3' 150 bp of exon 9A. In this study the distribution among chicken hematopoietic tissues of these two forms of alternative splicing was determined by Northern blot analysis using a probe specific for exon 9A. RNA species of 4.2 kilobases (kb) and 4.4 kb which contain exon 9A' or exon 9A, respectively, were detected in each tissue tested. Quantitative analysis of the major 4.0 kb c-myb species and the c-myb species containing exon 9A and exon 9A' revealed that cells from yolk sac contained both the highest absolute and the highest relative levels of alternatively spliced c-myb mRNA, presumably because of the preponderance of immature erythroid cells in these preparations.

Alternative splicing of the chicken c-myb exon 9A.

The c-myb gene products are thought to be regulators of cellular replication and of differentiation and heterogeneity may underlie their multiple functions. To investigate the possible existence of heterogeneity we have examined the chicken c-myb mRNAs by Northern blot analysis and polymerase chain reaction amplification of cDNAs (RT-PCR). Northern blot analysis with the c-myb cDNA clone pSG3, which contains the entire open reading frame (ORF) plus 500 base pairs of 3' untranslated sequences (Gerondakis & Bishop, 1986), and genomic probes revealed c-myb RNA species of 4.3 kb in addition to the major 4.0 kb species. The 4.3 kb c-myb RNA contained the alternatively spliced exon 9A which is highly conserved and has also been detected in a minor 4.3 kb alternatively spliced c-myb mRNA in murine and human cells. Sequencing of the avian exon 9A revealed 360 bp exon homologous to that found in murine and human mRNAs, which contains three highly conserved sequence regions shared by all three species. RT-PCR demonstrated usage of exon 9A in five hematopoietic tissues and revealed an additional splicing variant which used the 3' portion of exon 9A. Northern blot analysis using splice site-specific oligonucleotide probes spanning the two splice junctions between exon 9 and 9A revealed four additional c-myb RNAs of 4.4 kb, 2.2 kb, 2.0 kb and 1.4 kb.

Expression of MYB proteins in avian hematopoietic tissues.

MYB gene products are thought to be regulators of cellular replication and of differentiation. The major product of the avian MYB gene is a 75 kDa nuclear phosphoprotein which can activate transcription. A minor 89 kDa MYB protein of unknown function has also been described in murine and human cells. Additional heterogeneity at the level of MYB RNA which could affect the structure of MYB proteins has been described in several species. Such heterogeneity could explain the diverse effects of the MYB gene. To investigate the possible existence of heterogeneous and/or cell lineage-specific MYB proteins, five different avian hematopoietic tissues (bone marrow, bursa of Fabricius, embryonic spleen, thymus and yolk sac) were examined by immunoprecipitation with several MYB-specific antisera and SDS-PAGE analysis. In all five tissues there was a 75 kDa protein of uniform size which varied in abundance in a tissue-specific manner paralleling that observed for the 4.0 kb MYB RNA. A less abundant 89 kDa protein was also detected by several antisera in bone marrow, spleen, thymus and yolk sac but not in bursa. This 89 kDa MYB protein appears to be analogous to the 89 kDa MYB protein encoded by a minor but larger (360 nucleotides) MYB mRNA in murine and human cells. Immunoprecipitation of MYB proteins with an antiserum specific for exons 8 and 9 revealed a 74 kDa protein which co-precipitated and appeared to be complexed with p75 in normal hematopoietic cells and with the 48 kDa product of v-myb in leukemic cells.

Proto-oncogene expression in avian hematopoietic tissues.

Previous findings from this laboratory (Kim & Baluda, 1988) have shown that the proto-oncogenes ETS, FPS, MHT (RAF), MYC and REL are expressed in avian myeloblastosis virus (AMV)-transformed cells, whereas the MYB gene is repressed. In this study five different chicken hematopoietic tissues which contained varying concentrations of target cells for AMV transformation were analyzed to determine whether the expression of these proto-oncogenes resulted from, or was altered by, v-myb-induced leukemogenesis. Poly-A+ RNA from hematopoietic cells of 11-13 day yolk sac, 16 day embryonic spleen, 1 day post-hatch bursa of Fabricius, bone marrow and thymus, as well as from chicken embryonic fibroblasts (CEF) was examined by Northern blot analysis. All five proto-oncogenes were found to be expressed in the normal hematopoietic tissues. The ETS, MHT (RAF), MYC, and REL genes, but not FPS, were expressed in CEF. The expression of these five proto-oncogenes was not quantitatively or qualitatively altered in AMV-transformed myeloid cells as compared with their normal counterparts. While their expression is part of the hematopoietic phenotype of the target cells and as such is necessary for susceptibility to AMV transformation, it is not sufficient because thymocytes with a high level of expression are not transformed. This is in contrast to MYB expression, which is totally repressed in leukemic cells but probably not as a result of v-myb expression.

Hematopoietic lineage-specific heterogeneity in the 5'-terminal region of the chicken proto-myb transcript.

Comparison of the nucleotide sequence of the upstream c-myb exon UE3 with the sequences of a thymus c-myb cDNA and of a B-lymphoma c-myb cDNA suggested the existence of T- and B-cell-specific heterogeneity in the 5'-terminal region of the c-myb coding sequence. This possibility was investigated with T-cell-specific and B-cell-specific DNA probes in a Northern (RNA) blot analysis of mRNAs from different hematopoietic cell types and from chicken embryo fibroblasts. The hematopoietic tissues analyzed were bone marrow, bursa of Fabricius, and thymus from 1-day-old chicks, 13-day yolk sac, and spleen from 16-day embryos. At least three different c-myb mRNA species were found to have 5'-terminal heterogeneity that was specific for either B cells, T cells, or the other hematopoietic cells and chicken embryo fibroblasts. This lineage-specific heterogeneity in the c-myb transcript was found to be expressed in the bone marrow precursors of B and T cells before they migrated to their definitive differentiation sites. S1 nuclease protection analysis of the UE3 exon, part of which appeared to be coding sequences for thymic c-myb mRNA, revealed that this exon is utilized either in its entirety or partially in a cell-lineage-specific manner by all six tissues analyzed. Also, the 5'-terminal exon(s) present in the thymus cDNA was absent in c-myb mRNAs from the other cell types analyzed.

Proto-oncogene expression in chicken leukemic cells induced by avian myeloblastosis virus.

Sixteen proto-oncogenes which have generated retroviral oncogenes were tested for their expression in chicken leukemic cells induced by avian myeloblastosis virus (AMV) and five were found to be expressed (c-ets, c-fps, c-mht, c-myc, and c-rel). The size of the c-fps transcript (4.0 kb) was not in good agreement with the size (approximately 3.0 kb) previously reported but was uniform in the leukemic cells from 10 different chickens. The size of the other proto-oncogene transcripts appeared normal. The five expressed proto-oncogenes represent cellular genes involved in hematopoiesis. Interestingly the c-myb gene was not expressed in any of the leukemic cells despite its expression in the immature myeloid cells which are targets for AMV transformation. This could represent down regulation of c-myb by v-myb or a differentiation-related arrest of c-myb expression. The leukemic phenotype induced by v-myb may therefore become expressed at a stage of myeloid differentiation when c-myb expression is repressed.

Subnuclear associations of the v-myb oncogene product and actin are dependent on ionic strength during nuclear isolation.

The method used to isolate nuclei has a direct effect on the subnuclear association of the v-myb product, p48v-myb, and nuclear actin. Analysis of nuclei subjected to various isolation procedures showed that disruption of native nuclear structure during hypotonic treatment resulted in dissociation of p48v-myb from the nuclear matrix.

Expression of molecular clones of v-myb in avian and mammalian cells independently of transformation.

We demonstrated that molecular clones of the v-myb oncogene of avian myeloblastosis virus (AMV) can direct the synthesis of p48v-myb both in avian and mammalian cells which are not targets for transformation by AMV. To accomplish this, we constructed dominantly selectable avian leukosis virus derivatives which efficiently coexpress the protein products of the Tn5 neo gene and the v-myb oncogene. The use of chemically transformed QT6 quail cells for proviral DNA transfection or retroviral infection, followed by G418 selection, allowed the generation of cell lines which continuously produce both undeleted infectious neo-myb viral stocks and p48v-myb. The presence of a simian virus 40 origin of replication in the proviral plasmids also permitted high-level transient expression of p48v-myb in simian COS cells without intervening cycles of potentially mutagenic retroviral replication. These experiments establish that the previously reported DNA sequence of v-myb does in fact encode p48v-myb, the transforming protein of AMV.

Antibodies to the evolutionarily conserved amino-terminal region of the v-myb-encoded protein detect the c-myb protein in widely divergent metazoan species.

Antibodies directed against a bacterial fusion protein that contains the domain encoded by the highly evolutionarily conserved 5' one-third of the v-myb oncogene of avian myeloblastosis virus (AMV) detect the protein products of various members of the myb gene family. Immunoprecipitation or immunoblot analyses with these antibodies yielded the following information. First, the products of the v-myb oncogenes of AMV (p48v-myb) and of E26 virus (p135gag-myb-ets) contain this highly conserved amino acid sequence, as previously hypothesized. Second, p75c-myb, the product of the chicken c-myb protooncogene, also contains this protein domain. Third, these antibodies have identified the products of the human, murine, and Drosophila c-myb genes, which were all found to be nuclear proteins of Mr 75,000-80,000. The human c-myb protein product is present in immature cells of the erythroid, myeloid, and lymphoid lineages.

The myb oncogene.

The highly conserved, single copy c-myb gene has been independently transduced by two avian acute leukemia viruses, AMV and E26. This gene has also undergone insertional mutagenesis by non-acutely transforming murine leukemia viruses in a number of hematopoietic tumors. The common denominator of these retroviral activations of c-myb appears to be truncation of the normal coding region at either or both ends. The role of point mutations in myb-induced leukemogenesis is currently unknown. The products of the c-myb gene and its altered viral counterparts are nuclear proteins, a large fraction of which are associated with the nuclear matrix. In addition, the myb gene products have short half-lives and bind DNA in vitro. These features suggest that myb may act by regulating DNA replication or transcription. Consistent with this notion, the expression of c-myb is cell cycle dependent in several cell types. However, the abundant expression of c-myb in the thymus is not similarly regulated and may serve a different function. The expression of c-myb appears not to be limited to hematopoietic tissues as previously thought and the nature of the hematopoietic specificity of transformation by v-myb is not currently understood. Nevertheless, hematopoietic growth factors and their receptors appear to play an important role in such transformation. Two new experimental systems for studying myb have recently been described. First, the discovery of a myb-related gene in Drosophila should allow the application of powerful classical and molecular genetic approaches. The functional similarity of this distantly related gene to the much more closely related avian and mammalian myb genes is unknown. Second, recent studies of murine myb in normal and abnormal hematopoiesis offers several advantages relative to the avian system, such as in-bred animal strains, a wealth of specific cell-surface markers, and cloned hematopoietic growth factor and receptor genes. Isolation or construction of an acutely transforming murine myb retrovirus may thus be very useful. Several obvious goals for future research will be to define the function of myb proteins within the nucleus, to understand the regulation of myb expression during the cell cycle, to establish which molecular alterations are essential for converting c-myb into a transforming gene, and the determine the role of myb in human malignancies.

Nuclear compartmentalization of the v-myb oncogene product.

Nuclei obtained from chicken leukemic myeloblasts transformed by avian myeloblastosis virus were fractionated into various subnuclear compartments, which were then analyzed by specific immunoprecipitation for the presence of the leukemogenic product, p48v-myb, of the viral oncogene. In cells labeled for 30 or 60 min with L-[35S]methionine and in unlabeled exponentially dividing leukemic cells analyzed by Western blotting, p48v-myb was detected within the nucleoplasm (29 +/- 9% [standard deviation] of the total), chromatin (7 +/- 4%), and lamina-nuclear matrix (64 +/- 9%). Also, in myeloblasts analyzed by immunofluorescence during mitosis, p48v-myb appeared to be dispersed through the cell like the lamina-nuclear matrix complex. Strong attachment to the nuclear matrix-lamina complex suggests that p48v-myb may be involved in DNA replication or transcription or both.

Biologically active proviral clone of myeloblastosis-associated virus type 1: implications for the genesis of avian myeloblastosis virus.

A biologically active myeloblastosis-associated virus (MAV) provirus was cloned from a bacteriophage recombinant library constructed from leukemic chicken myeloblast DNA. The restriction endonuclease map of this clone was consistent with that of a type 1 MAV (MAV-1). Interference assays of virus recovered from cultured chicken embryo fibroblasts after DNA transfection established that the provirus was infectious and confirmed that it belonged to avian retrovirus subgroup A (type 1). Antipeptide antibodies raised against the env-encoded carboxyl terminus of p48myb, the transforming protein of avian myeloblastosis virus, specifically immunoprecipitated the gp37env from quail cells transfected with MAV-1 proviral DNA but not from cells infected with MAV-2. This suggests that MAV-1 rather than MAV-2 is the progenitor helper virus from which avian myeloblastosis virus arose by the transduction of cellular proto-oncogene sequences.