Uncovering residues that regulate cyclin D1 proteasomal degradation.

Cyclin D1 regulates G1 cell-cycle progression and is aberrantly expressed in carcinogenesis. Proteasomal degradation of cyclin D1 was highlighted as a cancer chemopreventive mechanism. To understand this mechanism better, residues responsible for degradation and ubiquitination of cyclin D1 were investigated. Eighteen lysines in cyclin D1 had single, double or multiple mutations engineered before transfection into BEAS-2B human bronchial epithelial (HBE) cells to evaluate stabilities after all-trans-retinoic acid (RA) or cycloheximide treatments. Specific mutations stabilized cyclin D1, including substitutions of lysines surrounding the cyclin box domain that inhibited RA-mediated degradation and extended the cyclin D1 half-life. Mutation of all cyclin D1 lysines blocked polyubiquitination. N-terminus (but not C-terminus) modification stabilized cyclin D1. Ubiquitination-resistant mutants preferentially localized cyclin D1 to the nucleus, directly implicating subcellular localization in regulating cyclin D1 degradation. Taken together, these findings uncover specific residues conferring ubiquitination of cyclin D1. These provide a mechanistic basis for proteasomal degradation of cyclin D1.

Transcriptional activation of the nuclear receptor corepressor RIP140 by retinoic acid: a potential negative-feedback regulatory mechanism.

Through the use of microarray analysis it was discovered that the nuclear receptor coregulator, receptor interacting protein 140 (RIP140), was induced early during all-trans retinoic acid (RA)-induced differentiation of human embryonal carcinoma cells. A rapid, fourfold induction of RIP140 mRNA was detected within 3 h of RA treatment in human embryonal carcinoma and MCF-7 human breast cancer cells. RIP140 protein levels were induced within 6 h of RA treatment. The RA induction of RIP140 mRNA did not require de novo protein synthesis, consistent with RIP140 being a direct transcriptional target of retinoid receptors. Promoter/enhancer elements directly upstream of the RIP140 coding region supported RA-induced transcription of a luciferase gene. In addition the ability of overexpressed RIP140 to repress ligand activated retinoid receptors was confirmed. The finding that RIP140 is a direct transcriptional target of RA is one of the first examples of acute transcriptional regulation of a nuclear receptor coactivator or corepressor. These data are consistent with a model by which RA induction of RIP140 supplies a negative feedback signal toward ligand-activated retinoid receptors.

Cyclin proteolysis as a retinoid cancer prevention mechanism.

The retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and prevention. Their biological effects are mediated through ligand-dependent interactions with retinoid receptors that associate with specific co-regulators. A better understanding of retinoid chemopreventive mechanisms is needed. Our prior work revealed that all-trans-retinoic acid (RA) prevented tobacco-specific carcinogenic transformation of cultured human bronchial epithelial cells. RA signaled G1 arrest that permitted repair of genomic DNA damage caused by these carcinogens. RA triggered G1 arrest at least partly through proteasome-dependent degradation of cyclin D1. Proteasomal inhibitors blocked RA-mediated cyclin D1 degradation. To confirm that a specific proteolysis pathway was induced by RA-treatment, a degradation assay was established using in vitro translated cyclin D1 and cellular extracts from RA-treated or untreated human bronchial epithelial cells. Incubation of RA-treated but not the control cellular extracts with in vitro translated cyclin D1 led to cyclin degradation. This degradation depended on the PEST domain of cyclin D1, implicating ubiquitination in this retinoid degradation. Retinoid receptor selective agonists demonstrated that retinoic acid receptor (RAR)beta and retinoid X receptor (RXR) but not RARalpha- or RARgamma-dependent pathways signaled this cyclin degradation. Findings were extended to the NT2/D1 human embryonal carcinoma differentiation model where a similar pathway was activated by RA-treatment. To determine whether G1 cyclins were involved directly in bronchial preneoplasia, immunohistochemical expression profiles for cyclins D1 and E were examined. Aberrant expression of these cyclins was frequent in bronchial preneoplasia. Taken together, these findings indicate that ubiquitin-dependent proteolysis of G1 cyclins is a retinoid chemoprevention mechanism. Whether the retinoids represent the optimal agents to activate this pathway is the subject of ongoing work. These findings provide a rationale for combining the retinoids in chemoprevention trials with other agents that do not activate this proteolysis pathway. What is now known about the retinoids as cancer prevention agents will be reviewed. Emphasis is placed on retinoid effects on cell cycle progression at G1.

Retinoic acid promotes ubiquitination and proteolysis of cyclin D1 during induced tumor cell differentiation.

Mechanisms by which differentiation programs engage the cell cycle are poorly understood. This study demonstrates that retinoids promote ubiquitination and degradation of cyclin D1 during retinoid-induced differentiation of human embryonal carcinoma cells. In response to all-trans-retinoic acid (RA) treatment, the human embryonal carcinoma cell line NT2/D1 exhibits a progressive decline in cyclin D1 expression beginning when the cells are committed to differentiate, but before onset of terminal neuronal differentiation. The decrease in cyclin D1 protein is tightly associated with the accumulation of hypophosphorylated forms of the retinoblastoma protein and G(1) arrest. In contrast, retinoic acid receptor gamma-deficient NT2/D1-R1 cells do not growth-arrest or accumulate in G(1) and have persistent cyclin D1 overexpression despite RA treatment. Notably, stable transfection of retinoic acid receptor gamma restores RA-mediated growth suppression and differentiation to NT2/D1-R1 cells and restores the decline of cyclin D1. The proteasome inhibitor LLnL blocks this RA-mediated decline in cyclin D1. RA treatment markedly accelerates ubiquitination of wild-type cyclin D1, but not a cyclin D1 (T286A) mutant. Transient expression of cyclin D1 (T286A) in NT2/D1 cells blocks RA-mediated transcriptional decline of a differentiation-sensitive reporter plasmid and represses induction of immunophenotypic neuronal markers. Taken together, these findings strongly implicate RA-mediated degradation of cyclin D1 as a means of coupling induced differentiation and cell cycle control of human embryonal carcinoma cells.

Transcription of the human folylpoly-gamma-glutamate synthetase gene.

In mammals, folylpoly-gamma-glutamate synthetase (FPGS) activity is found in any cell undergoing sustained proliferative phases, but this enzyme also displays a tissue-specific pattern of expression in differentiated tissues. It is now reported that the steady state levels of FPGS mRNA in normal and neoplastic cells reflect these patterns, supporting the concept that the control mechanisms underlying this distribution are transcriptional. To initiate an understanding of these interacting levels of control, we have determined the position and properties of the minimal FPGS promoter controlling transcription of the FPGS gene in human CEM leukemia cells, a line which expresses high levels of this enzyme and its mRNA. The TATA-less region immediately upstream of the major transcriptional start site previously mapped in human tumor cells, which includes several GC- and Y-boxes, functioned as a remarkably efficient promoter when used to drive expression of a luciferase reporter in transient expression studies in CEM cells. The minimal region of the FPGS promoter required for maximal transcriptional activation in CEM cells included the 80 base pairs over which the multiple transcriptional start sites were located, and the 43 base pairs immediately upstream. DNase I footprint analysis detected the binding of Sp1 at all seven of the consensus sites within the probe used, two of which are contained within the minimal promoter region. The several Sp1 sites immediately upstream of the first major transcriptional start activated transcription in Drosophila cells when cotransfected with an Sp1 construct, including those in the region which functioned as a minimal promoter in CEM cells. An additional region of the minimal promoter, situated between the two translational start codons of the FPGS gene, was bound by protein(s) from HeLa cell nuclear extracts. We conclude that transcription of the FPGS gene in CEM cells involves transactivation events over a limited upstream DNA sequence and that the FPGS promoter used in proliferating human leukemic cells has strong similarity to other TATA-less promoters that utilize tandem, closely spaced Sp1 sites to initiate transcription.

Comparison of methotrexate polyglutamylation in L1210 leukemia cells when influx is mediated by the reduced folate carrier or the folate receptor. Lack of evidence for influx route-specific effects.

We previously described a methotrexate-resistant L1210 cell line (MTXrA) that lacks a functional reduced folate carrier and does not appreciably express the folate receptor. In the present study, we utilized MTXrA cell lines stably transfected with cDNAs encoding either the folate receptor or the reduced folate carrier to investigate the influence of the route of folate influx on the rate and extent of methotrexate polyglutamylation. At an extracellular methotrexate concentration of 0.1 microM, influx in the folate receptor transfectant (MTXrA-TF1) and in the reduced folate carrier transfectant (MTXrA-R1) was equal and methotrexate polyglutamates accumulated at an identical rate, but the onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (approxmately 3 hr). The onset of polyglutamate formation was immediate and identical among the lines in cells pretreated with the lipophilic dihydrofolate reductase inhibitor trimetrexate to block methotrexate binding to dihydrofolate reductase. The spectra of individual methotrexate polyglutamates that accumulated were similar, with the tetraglutamate present as the predominant form. A 100-fold higher methotrexate concentration was required to detect methotrexate uptake and polyglutamylation in the transport defective parent MTXrA line, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the reduced folate carrier achieve nearly identical rates of polyglutamylation despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglutamate synthetase is unlikely to play a role in tetrahydrofolate metabolism. This study supports the notion that it is the intracellular concentration of methotrexate achieved within the cell that drives polyglutamylation irrespective of its route of entry.

Structural organization of the human folypoly-gamma-glutamate synthetase gene: evidence for a single genomic locus.

The cytotoxicity, and probably the selectivity, of folate antimetabolites depend upon the expression of the enzyme folylpoly-gamma-glutamate synthetase in tumor cells. Evidence for the existence of multiple forms of this enzyme and the need to define the control mechanisms determinant of expression levels in normal and neoplastic cells has focused attention on the gene(s) encoding these forms. The organization of the genomic locus for the human folylpoly-gamma-glutamate synthetase (FPGS) gene has been determined. The complete 2256 nucleotides of cDNA for the 5'-untranslated region, mitochondrial leader sequence, coding region, and 3'-untranslated region were distributed on 15 exons stretching over 11.2 kb of genomic DNA. All of the restriction fragments found in diploid human genomic DNA could be accounted for by fragments contained on the isolated genomic clones. Likewise, Southern analysis of the transfected human genomic DNA that complemented the FPGS- phenotype of a hamster cell line indicated that the same gene had been integrated in all of three independently derived transfectants. We conclude that the genomic locus that we now report appears to be the only gene encoding FPGS-related sequences in the human complement.

Molecular characterisation of two cell lines selected for resistance to the folate-based thymidylate synthase inhibitor, ZD1694.

Resistance to anti-cancer drugs has proved to be a major barrier in the clinical management of neoplastic disease. We have investigated the mechanistic basis for resistance to folate-based thymidylate synthase (TS) inhibitors using two cell lines selected for resistance to ZD1694 (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N -methylamino]-2 - thenoyl)-L-glutamic acid), a drug currently in phase III clinical trial. The degree of resistance was > 20,000 for the human lymphoblastoid cell line W1L2:R and approximately 14 for the ovarian carcinoma cell line CH1:R. In both cases resistance was associated with increased TS activity. The W1L2:R cell line had an approximately 100-fold increase in TS gene copy number and mRNA levels and a 500- to 1000-fold increase in enzyme levels determined using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern and Western blotting. The CH1:R cell line had an approximately 2- to 2.5-fold increase in TS gene copy number, mRNA and protein levels. In both cell lines the fold resistance determined was significantly higher than the fold increase in target enzyme DNA, mRNA or protein levels. Small changes in TS levels may therefore translate to clinically significant alterations in drug sensitivity.

Upstream organization of and multiple transcripts from the human folylpoly-gamma-glutamate synthetase gene.

Folylpoly-gamma-glutamate synthetase (FPGS) is essential for the survival of proliferating mammalian cells and central to the action of all "classical" folate antimetabolites. We report the isolation of cDNAs corresponding to the 5' ends of FPGS mRNA from both human and hamster cells which include a start codon upstream of and in-frame with the AUG in the previously reported FPGS open reading frame. The predicted hamster and human amino-terminal extension peptides have features consistent with a mitochondrial targeting sequence. Ribonuclease protection and 5'-rapid amplification of cDNA ends assays indicated multiple transcriptional start sites consistent with the sequence of the promoter region of this gene, which was highly GC-rich and did not contain TATA or CCAAT elements. These start sites would generate two classes of transcripts, one including the upstream AUG and one in which only the downstream AUG would be available for translation initiation. Transfection of the full length human cDNA into cells lacking FPGS restored their ability to grow in the absence of glycine, a product of mitochondrial folate metabolism, as well as of thymidine and purines. Therefore, we propose that the mitochondrial and cytosolic forms of FPGS are derived from the same gene, arising from the use of the two different translation initiation codons, and that the translation products differ by the presence of a 42-residue amino-terminal mitochondrial leader peptide.