Identification of serum-inducible genes: different patterns of gene regulation during G0-->S and G1-->S progression.
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Amino Acid Sequence↗
Biomedical subjects
F C Lucibello
Publications and source records attributed to F C Lucibello.
Inducible acceleration of G1 progression through tetracycline-regulated expression of human cyclin E.
Cyclin E is a cell cycle-regulated protein that activates the cdc2-related protein kinases cdk2 shortly before S-phase entry. In order to analyse the biological role of cyclin E, we have generated HeLa cells that allow the conditional expression of ectopic human cyclin E. In these cells, a cyclin E cDNA is under the control of a bacterial tetracycline repressor-VP16 activator hybrid protein. In the absence of tetracycline, the endogenous gene becomes activated and leads to the synthesis of elevated levels of cyclin E. Concomitant with this increase in cyclin E expression we show by a combined time-lapse video recording/5-bromo-deoxyuridine labelling procedure a significant acceleration of G1 transition by approximately 1.5 hours. This observation is consistent with the idea that cyclin E is a rate-limiting factor with respect to the control of G1-->S transition. The experimental system described here should also prove useful to address the function of cyclin E in further detail.
Cyclins↗
Oncogenic activity of cyclin D1 revealed through cooperation with Ha-ras: link between cell cycle control and malignant transformation.
Circumstantial evidence implicates the putative cell cycle regulator cyclin D1 in the process of malignant transformation. Overexpression of cyclin D1 is observed in mammary carcinomas as a result of gene amplification and in parathyroid adenomas and centrocytic B-cell lymphomas as a consequence of chromosomal rearrangements and juxtaposition of the cyclin D1 gene to strong transcriptional control elements. These findings suggest that deregulation of cyclin D1 expression may contribute to malignant transformation in these tumours. To date, however, an oncogenic potential of cyclin D1 has not been demonstrated and the mechanism of its oncogenic activation remains obscure although overexpression of the wild-type protein is likely. We report here that the overexpression of cyclin D1 induces transformation in primary rat embryo fibroblasts in cooperation with activated Ha-ras. Cyclin D1/Ha-ras transformed cells are immortalized, show anchorage independence and give rise to fibrosarcomas in nude mice. Our data directly demonstrate that cyclin D1 is a proto-oncogene that can be activated by transcriptional deregulation. Its previously demonstrated ability to interact with putative cell cycle regulators suggests that cyclin D1 defines a new class of proto-oncogenes.
Animals↗
Signals and genes in the control of cell-cycle progression.
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Animals↗
Human cyclin D1 encodes a labile nuclear protein whose synthesis is directly induced by growth factors and suppressed by cyclic AMP.
We show that the cyclin D1 gene is regulated by a variety of growth factors in human diploid fibroblasts (WI-38). Expression of cyclin D1 mRNA is low in quiescent WI-38 cells and reaches a maximum around 10 hours after serum stimulation, i.e. approximately 8 hours prior to the onset of DNA synthesis. A cyclin D1-specific antiserum raised against a bacterially expressed fusion protein detected a 39 kDa polypeptide in WI-38 cells. In agreement with the RNA expression data, cyclin D1 protein synthesis is also serum-inducible, reaching a maximum around 9 hours post-stimulation. The results obtained by pulse-chase experiments, cell fractionation and immunostaining techniques strongly suggest that cyclin D1 is a labile protein (t1/2 approximately 38 min), which is located in the nucleus. Cyclin D1 is directly induced by growth factors, i.e. in the presence of cycloheximide, and its expression does not significantly fluctuate during the cell cycle in synchronized cells. Cyclin D1 therefore fundamentally differs from "classical" cyclins, such as the mitotic cyclin B, whose expression is clearly cell cycle-dependent. Cyclin D1 may rather establish a direct link between growth control mechanisms and the cell cycle. Interestingly, cyclin D1 expression is stimulated by the protein kinase C activator TPA, but suppressed by dibutyryl-cAMP and the adenylate cyclase inducer forskolin, pointing to multiple regulatory pathways controlling cyclin D1 expression.
Base Sequence↗
Deregulation of cyclins D1 and E and suppression of cdk2 and cdk4 in senescent human fibroblasts.
The state of cellular senescence is characterised by an irreversible arrest in the G1 phase of the cell cycle. It has previously been shown that three cell cycle genes, cyclin A, cyclin B and cdc2, are not expressed in senescent human fibroblasts. All three gene products have functions after S-phase entry, so that their suppression cannot explain the irreversible G1 arrest. Here, we report that the abundance of transcripts from two other cell cycle genes, cdk2 and cdk4, thought to act during G1-->S progression, is significantly diminished in senescent cells of the diploid human fibroblast line WI-38. Surprisingly, two other cyclins, D1 and E, behave in a completely different way, in that their expression is elevated in senescent cells, especially under conditions of serum starvation. Both the synthesis and the steady-state level of cyclin D1 protein were also found to be markedly higher in senescent cells (3- to 6-fold). Cyclins D1 and E are thus the first genes shown to be overexpressed or deregulated in senescent cells. It is tempting to speculate that this deregulation may be due to the absence, in senescent cells, of a regulatory loop that would normally control their expression. This is supported by our finding that cyclin E-associated kinase activity in senescent cells is reduced approx. 14-fold. Our data also suggest that the deregulated expression of cyclin D1 and E is not sufficient to drive senescent cells into DNA replication.
Base Sequence↗
Suppression of the growth factor-mediated induction of c-fos and down-modulation of AP-1-binding activity are not required for cellular senescence.
It has been suggested that the impaired response of the c-fos gene to serum growth factors and the concomitant loss of AP-1 activity may be a crucial step in the process of cellular senescence. In the present study, we provide evidence arguing against such a conclusion. Data obtained in five independent experiments showed that both c-fos RNA and protein expression were similar in 'young' and in senescent serum-stimulated WI-38 cells, suggesting that the previously reported suppression of c-fos induction is not an obligatory event in the process of cellular senescence. Likewise, expression of fra-1, c-jun and junB continued to be high in serum-stimulated senescent cells, while induction of fosB was reduced approximately fivefold. Among all genes tested fosB thus seems to be the most suitable marker for the detection of senescent cells. Stimulated senescent cells showed only a approximately twofold reduction of AP-1-binding activity, and senescent cells continuously exposed to serum exhibited normal AP-1-binding activity. These observations support the conclusion that a down-modulation of AP-1 is not crucial for human fibroblasts to enter the senescent state.
Cell Line↗
Transcription factor encoding oncogenes.
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Animals↗
Inhibition of Fos- and Ras-induced transformation by mutant Fos proteins with structural alterations in functionally different domains.
We show that transformation-defective Fos proteins lacking either a functional leucine zipper (mutants L345 and J/R510s) or the 110 amino-terminal amino acids (mutant BR800) inhibit the induction of morphological transformation by v-Fos. Both types of mutants specifically repress transformation without any significant effect on cell proliferation. In contrast, several transformation-defective Fos mutants with structural alterations in the acidic region or the right half of the adjacent basic DNA contact site do not show any inhibition of transformation. This result, taken together with the repression of transformation by the leucine zipper-deficient mutants L345 and J/R510s, indicates that the interaction of Fos with proteins other than Jun is necessary for transformation. The leucine zipper-deficient mutants also inhibit Fos-mediated activation of AP-1-dependent transcription. This suggests that their inhibitory effect on transformation may at least in part be the result of the squelching of proteins other than Jun family members that are required for Fos-mediated transactivation. All three mutants were also found to inhibit transformation by the point-mutated H-ras oncogene from EJ carcinoma cells and to trigger a partial reversion of the transformed phenotype of Ras-transformed fibroblasts. These findings support the conclusion that Ras-induced transformation involves signal transduction pathways inducing the c-fos gene.
Cell Division↗
Multiple interdependent regulatory sites in the mouse c-fos promoter determine basal level transcription: cell type-specific effects.
Although the induction of the mouse c-fos promoter by growth factors and specific signal transduction pathways has been analyzed in some detail, the mechanisms involved in the control of basal level transcription remain largely elusive. In this study, we present evidence for the existence of at least 9 different elements, located between the putative TATA box and position -610, that figure in basal level transcription and represent protein binding sites in different cell types. A major regulatory site in F9END, NIH3T3 and HeLa cells is the CRE around position -60. Other sites, including the SRE, a NF1 site around position -165, a novel site downstream of the SRE and three new sites upstream of the SRE play different cell type-specific roles. In addition, we have identified two regions upstream of the SRE, which seem to have cell type-specific negative regulatory effects. We also find that the precise function of several of these sites depends on the presence or absence of other elements, indicating some form of interaction between different regulatory sites. Finally, we present evidence, that the block of c-fos transcription in F9EC cells is due to the lack of transregulatory proteins, which are induced during retinoic acid mediated differentiation.
Animals↗
Alternative splicing of fosB transcripts results in differentially expressed mRNAs encoding functionally antagonistic proteins.
We show that serum-stimulated fibroblasts transiently express two different forms of fosB mRNA, which are generated by alternative splicing of the transcript from a single gene. In addition to the known long form (fosB-L), encoding a protein of 338 amino acids (FosB-L), a second shorter form (fosB-S) with a deletion of 140 bp was detected. This deletion creates a stop codon 3' to the leucine repeat, giving rise to a protein of 237 amino acids (FosB-S) lacking the carboxyl terminus of FosB-L. Only the long FosB form efficiently induces transformation in mouse and rat fibroblast cell lines and trans-represses the c-fos promoter. Both of these functions are suppressed by coexpressed FosB-S. Upon serum stimulation, maximum expression of the oncogenic fosB-L form precedes the expression of the antagonistic fosB-S form, indicating a new mechanisms regulating the action of members of the Fos family. However, FosB-L and FosB-S do not differ in all trans-regulatory properties: Trans-activation of a 5x TRE-CAT reporter construct in HeLa and NIH-3T3 cells was found with both FosB forms. These observations suggest a correlation between fosB-induced transformation and trans-repression, thus pointing to different mechanisms involved in transformation by fosB and c-fos/v-fos.
Animals↗
Multiple regions of v-Fos protein involved in the activation of AP1-dependent transcription: is trans-activation crucial for transformation?
We show that trans-activation by v-Fos requires several functionally separable regions, including the leucine repeat, the basic DNA-binding region, a directly adjacent acidic cluster, and additional flanking sequences. Structural alterations in the flanking regions are in part responsible for the greater trans-activating potential of the fos gene product of the Finkel-Biskis-Reilly mouse osteosarcoma virus, FBR-MuSV. A point mutation in the acidic cluster, which is known to activate the immortalizing potential of Fos, leads to a significant increase in trans-activation. However, comparison of the trans-activating and transforming properties of mutant Fos proteins suggests that functions other than trans-activation are involved in the induction of transformation.
Cell Transformation, Neoplastic↗
Proto-oncogenes encoding transcriptional regulators: unravelling the mechanisms of oncogenic conversion.
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Animals↗
Mutual transrepression of Fos and the glucocorticoid receptor: involvement of a functional domain in Fos which is absent in FosB.
In this study, we show that Fos protein can repress transactivation by the glucocorticoid receptor (GR). In addition, we demonstrate that GR is capable of inhibiting, in a hormone-dependent fashion, Fos-mediated transactivation of AP-1 dependent transcription. Moreover, repression of the serum response element by Fos is abolished by the GR in the presence of hormone. Transrepression of glucocorticoid mediated induction involves a region of Fos, located between amino acids 40 and 111, to which no function has been previously assigned, and which is poorly conserved among Fos, FosB and Fra-1. In agreement with this finding, FosB is not capable of transrepressing GR activation of transcription, representing the first functional difference between Fos and FosB. We have mapped the domain of the GR which is required for repression of AP-1 dependent transcription, to the region of central DNA binding domain. Our results suggest that Fos and the GR may form transcriptionally inactive complexes and point to a regulatory interrelationship between different signal transduction pathways.
Amino Acid Sequence↗
trans-repression of the mouse c-fos promoter: a novel mechanism of Fos-mediated trans-regulation.
Fos protein can trans-activate AP-1-dependent gene expression and trans-repress the c-fos promoter. Although we find that trans-repression is enhanced by coexpression of c-Jun, it does not require any of the AP-1 or ATF sites in the mouse c-fos promoter. A major target for repression is the serum response element (SRE). Fos mutants with an impaired leucine zipper are defective in trans-repression and transformation, suggesting that these functions involve the formation of Fos protein complexes. In contrast, mutations that abolish DNA binding of Fos enhance trans-repression but destroy the transforming potential of Fos. In addition, v-Fos protein efficiently transforms but is unable to trans-repress. These findings point to different mechanisms involved in trans-activation and trans-repression and suggest that trans-repression of the type described here is neither sufficient nor required for Fos-induced transformation.
Animals↗
A rapid micro method for counting cells "in situ" using a fluorogenic alkaline phosphatase enzyme assay.
A new method has been developed to count cells "in situ", based on a fluorogenic enzyme assay that measures the activity of alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship did not change with time in culture. The alkaline phosphatase assay (ALP assay) was able to estimate relative cell numbers over a range from about 10(4) to 5 X 10(5) for many cell types, including Hep-2, a derivative of HeLa, several human colorectal cell lines SW1222, SW837, LS174T and HT29, a normal human diploid cell strain MRC5 and a rodent line NIH-3T3. The ALP assay is rapid and efficient, making it a useful method for studying growth assays.
Alkaline Phosphatase↗
Localization of the gene for familial adenomatous polyposis on chromosome 5.
Colorectal cancer is the second most common cancer in the United Kingdom and other developed countries in the West. Although it is usually not familial, there is a rare dominantly inherited susceptibility to colon cancer, familial adenomatous polyposis (FAP; also often previously called familial polyposis coli). During adolescence affected individuals develop from a few hundred to over a thousand adenomatous polyps in their large bowel. These are sufficiently likely to give rise to adenocarcinomas to make prophylactic removal of the colon usual in diagnosed FAP individuals. Adenomas may occur elsewhere in the gastrointestinal tract and the condition is often associated with other extracolonic lesions, such as epidermoid cysts, jaw osteomata and fibrous desmoid tumours. Adenomata have been suggested to be precancerous states for most colorectal tumours. Knudson has suggested that the mutation for a dominantly inherited cancer susceptibility may be the first step in a recessive change in the tumour cells, and that the same gene may be involved in both familial and non-familial cases of a given tumour. Following up a case report of an interstitial deletion of chromosome 5 in a mentally retarded individual with multiple developmental abnormalities and FAP, we have now shown that the FAP gene is on chromosome 5, most probably near bands 5q21-q22.
Adenocarcinoma↗
Chromosome 5 allele loss in human colorectal carcinomas.
That the sporadic and inherited forms of a particular cancer could both result from mutations in the same gene was first proposed by Knudson. He further proposed that these mutations act recessively at the cellular level, and that both copies of the gene must be lost for the cancer to develop. In sporadic cases both events occur somatically whereas in dominant familial cases susceptibility is inherited through a germline mutation and the cancer develops after a somatic change in the homologous allele. This model has since been substantiated in the case of retinoblastoma, Wilms tumour, acoustic neuroma and several other tumours, in which loss of heterozygosity was shown in tumour material compared to normal tissue from the same patient. The dominantly inherited disorder, familial adenomatous polyposis (FAP, also called familial polyposis coli), which gives rise to multiple adenomatous polyps in the colon that have a relatively high probability of progressing to a malignant adenocarcinoma, provides a basis for studying recessive genes in the far more common colorectal carcinomas using this approach. Following a clue as to the location of the FAP gene given by a case report of an individual with an interstitial deletion of chromosome 5q, who had FAP and multiple developmental abnormalities, we have examined sporadic colorectal adenocarcinomas for loss of alleles on chromosome 5. Using a highly polymorphic 'minisatellite' probe which maps to chromosome 5q we have shown that at least 20% of this highly heterogeneous set of tumours lose one of the alleles present in matched normal tissue. This parallels the assignment of the FAP gene to chromosome 5 (see accompanying paper) and suggests that becoming recessive for this gene may be a critical step in the progression of a relatively high proportion of colorectal cancers.
Adenocarcinoma↗