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K Keyomarsi

Publications and source records attributed to K Keyomarsi.

At least 37 records · Page 2Linked to original sources
PubMed1997

The role of cyclin E in cell proliferation, development and cancer.

Normal cell proliferation is under strict regulation governed by checkpoints located at distinct points in the cell cycle. The deregulation of these checkpoint events and the molecules associated with them may transform a normal cell into a cancer cell. One of these checkpoints whose deregulation results in transformation occurs at the Restriction point, near the G1/S boundary. The periodic appearance of one of the recently identified regulatory cyclins, cyclin E, coincides precisely with the timing of the Restriction point. The deregulation in the expression and activity of cyclin E has been associated with a number of cancers and is thought to be involved in the process of oncogenesis. In this chapter, we summarise the current knowledge on the regulation and apparent function of cyclin E in normal proliferating cells and in developing tissue and alterations of these processes in cancer.

Animals↗
PubMed1996

Cyclin E, a redundant cyclin in breast cancer.

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G1/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.

Animals↗
PubMed1996

Neu differentiation factor (Heregulin) activates a p53-dependent pathway in cancer cells.

Previously we reported that neu differentiation factor (NDF)/heregulin (HRG) elevates tyrosine phosphorylation of its receptors erbB-3, erbB-4, and erbB-2 (through heterodimer formation). We also showed that both NDF/HRG and antibodies to erbB-2 can arrest growth and induce differentiation in breast cancer cells. In this study, we report on the mechanism of NDF/HRG-induced cellular effects. We show that NDF/HRG and antibodies to erbB-2 receptors up-regulate expression of p53 by stabilizing the protein. This is accompanied by up-regulation of the p53 inducible gene, p21CIP1/WAF1, in a variety of cell lines: MCF7 and their derivatives (MCF7/HER2, MN1 and MCF-7-puro), ZR75T and LnCap cells. The induction of p21 is further enhanced when cells are treated with both NDF/HRG and DNA-damaging chemotherapeutic agents (i.e. doxorubicin). The NDF/HRG mediated induction of p21 is dependent on wildtype p53, as it fails to occur in cells expressing dominant negative p53 (MDD2). Furthermore, p21 induction is capable of inactivating cdk2 complexes as measured by Histone H1 phosphorylation assays. Finally, we show that in primary cultures of breast and other cancers, p21 is significantly induced in response to NDF/HRG treatment. Collectively, these observations suggest that the mechanism of breast cancer cell growth inhibition and differentiation via erbB receptors activation is through a p53-mediated pathway.

Antibodies, Monoclonal↗
PubMed1996

Expression of cytochromes P450 in human breast tissue and tumors.

In an effort to determine which members of the cytochrome P450 (CYP) superfamily are expressed in human breast tissue and tumors, RNA-polymerase chain reaction studies have been undertaken. Detection of expressed CYP mRNAs identifies those forms of the enzyme that are capable of expression in breast tissue, and provides insight into the potential for in situ xenobiotic and therapeutic drug metabolism. CYP1A1 mRNA was present in (5/11) breast tissues and (6/13) tumors. When normal and tumor tissues were from the same individuals, higher amplification occurred in normal tissues. CYP1B1 mRNA was present in all but one tissue, and CYP2C mRNA forms were present in all of the tissues. CYP3A4 mRNA was present in (8/11) normal breast tissues and (2/13) tumor tissues, and CYP3A5 mRNA was present in (9/11) normal tissues and (2/13) tumor tissues. The expression of the CYP3A mRNA forms was not coincident, suggesting differential regulation. CYP2D6 mRNA was present in (10/11) normal breast tissue and (10/13) tumors. Two splice variants of CYP2D6 mRNA were also detected; one with a 207 bp intron spliced in was detected in all of the normal tissue samples and (11/13) tumors, whereas another (which lacks a 3'-portion of exon 6) was detected in (9/11) normal breast tissues and (7/13) tumors. Thus, examples of each of the xenobiotic-metabolizing CYP1, CYP2, and CYP3 subfamilies were detected in low levels in human normal breast tissue and tumors. The machinery for possible in situ bioactivation of xenobiotics and modification of therapeutic drugs is thus present in human breast tissue.

Breast↗
PubMed1995

Deregulation of cyclin E in breast cancer.

Cyclin E, a regulatory subunit of cyclin dependent kinase-2, is thought to be rate limiting for the G1/S transition during the mammalian cell cycle. Previously, we showed severe alterations in cyclin E protein expression in human mammary epithelial cell lines and in surgical material obtained from patients with various malignancies. To understand the functional basis of these alterations we analyse here the regulation of cyclin E in breast cancer cells. We find that while cyclin E protein and its associated kinase activity in normal cells are cell cycle regulated, in tumor cells it remains in an active complex throughout the cell cycle. We also analysed cyclin E for possible deletions which could result in its constitutive function and found two novel truncated variants in its coding region. These variant forms of cyclin E were detected in several normal and tumor cell lines and tissue specimens. However, Western blot analysis indicated that only the multiple isoforms of cyclin E protein were expressed in tumor but not the normal tissue specimen, suggesting post transcriptional regulation of cyclin E. Lastly, in vitro analyses indicated that these truncated variant forms of cyclin E are biochemically active in their ability to phosphorylate histone H1. Collectively these observations suggest the presence of more than one form of cyclin E mRNA in all cells, normal and tumor. Once translated in tumor cells, the protein products of these truncated forms could give rise to a constitutively active form of cyclin E containing complexes.

Base Sequence↗
PubMed1995

Inhibition of cyclin-dependent kinases by p21.

p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression.

Binding, Competitive↗
PubMed1994

Synthesis and biological activity of N omega-hemiphthaloyl-alpha,omega- diaminoalkanoic acid analogues of aminopterin and 3',5-dichloroaminopterin.

Analogues of N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-ornithine (PT523) with 3',5'-dichloro substitution in the p-aminobenzoyl moiety or with one less or one more CH2 group in the amino acid moiety were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) activity and cell growth. Replacement of L-ornithine in PT523 by L-2,4-diaminobutanoic acid or L-lysine did not decrease binding to human recombinant DHFR but resulted in some loss of activity against SCC25 human and SCC VII murine squamous cell carcinoma and against MCF-7 human breast carcinoma in culture. PT523 was several times more potent than methotrexate (MTX), aminopterin (AMT), or trimetrexate (TMQ). 3',5'-Dichloro substitution did not decrease either DHFR binding or cytotoxicity. A new synthetic route to PT523 from 2,4-diamino-6-(hydroxymethyl)pteridine and methyl N alpha-(4-aminobenzoyl)-N delta-phthaloyl-L-ornithinate was investigated but was not found superior to previously described methods. In comparative experiments on the ability of PT523 and MTX to competitively inhibit the influx of (6R)-5,10-dideazatetra-hydrofolate (DDATHF, lometrexol), used here as a surrogate for MTX and reduced folates, the Ki of PT523 was lower than that of MTX in both wild-type CCRF-CEM human leukemic lymphoblasts and the transport- and polyglutamylation-defective subline CEM/MTX. The CCRF-CEM cells were 10-fold more sensitive to PT523 than to MTX, whereas the CEM/MTX cells were 240-fold more sensitive. However, in contrast to other MTX-resistant cells where collateral sensitivity to PT523 has been seen. CEM/MTX cells still showed substantial cross resistance to PT523 which may reflect an unusual heightened ability to utilize exogenous folic acid. The good correlation observed with both cell lines between the cytotoxicity of PT523 and MTX and the ability to inhibit DDATHF influx supported the view that PT523 and MTX share, at least in part, a common protein carrier for membrane transport.

Aminopterin↗
PubMed1994

Cyclin E, a potential prognostic marker for breast cancer.

A fundamental cause of cancer is changed properties of genetic material, which may deregulate normal development of the tissue or provide selective growth advantage to the tumor cell. This deregulation of cell proliferation results from altered production of a handful of proteins that play key roles in progression through the eukaryotic cell cycle. Some of these proteins include tumor suppressor genes or oncogenes. However, no one general change or alteration of a critical gene has yet been found in all cancers. Using surgical material obtained from patients with various malignancies, we show that breast cancers and other solid tumors, as well as malignant lymphocytes from patients with lymphatic leukemia, show severe quantitative and qualitative alterations in cyclin E protein production independent of the S-phase fraction of the samples. Hence, these alterations represent a true difference between normal versus tumor tissue. In addition, in breast cancer, the alterations in cyclin E expression become progressively worse with increasing stage and grade of the tumor, suggesting its potential use as a new prognostic marker.

Biomarkers, Tumor↗
PubMed1993

The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.

The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.

Amino Acid Sequence↗
PubMed1993

The thymidylate synthase inhibitor, ICI D1694, overcomes translational detainment of the enzyme.

We have investigated the mechanism of inactivation of thymidylate synthase (TS) by ICI D1694 (a folate-based quinazoline) in normal versus tumor-derived human mammary epithelial cells. ICI D1694 is a very potent cytotoxic agent against these cells with IC50 values of 1-2 nM. Its growth inhibitory activity was completely reversed by the addition of thymidine, confirming that TS is its sole target in these cells. Remarkably, TS protein levels rose by 10-40-fold following treatment with ICI D1694, depending on cell type, while TS mRNA levels remained constant. The mechanism appears to be a release of "detainment" of TS translation, since addition of cycloheximide, a translational inhibitor, blocked the TS protein levels from rising. But coadministration of 5,6-dichlorobenzimidazole, a transcriptional inhibitor, did not overcome protein accumulation, nor did thymidine which overcomes growth inhibition by ICI D1694. 5,10-Methylenetetrahydrofolate (via folinic acid), however, did block the effects of ICI D1694, showing that the drug has its effect upon both detainment and enzyme inhibition by binding to the folate substrate site of TS. In addition, in the presence of ICI D1694, TS protein was no longer cell cycle-regulated as evident by its constitutive expression in synchronized cells. This accumulation and constitutive expression of TS induced by D1694 should increase drug resistance under a clinical setting. We suggest that an ideal inhibitor of TS would target the TS allosteric site that binds to TS mRNA, responsible for specific translation of the protein, thereby complimenting inactivation of the enzyme.

Cell Cycle↗
PubMed1993

Redundant cyclin overexpression and gene amplification in breast cancer cells.

Cyclins are prime cell cycle regulators and are central to the control of major check points in eukaryotic cells. The aberrant expressions of two cyclins (i.e., cyclins A and D1) have been observed in some cancers, suggesting they may be involved in loss of growth control. However, in spite of these occasional changes involving only two cyclins, there are no clear connections between general derangements of other cyclins or their dependent kinases in a single tumor type. We detected general cyclin overexpression in 3 of 3 breast tumor tissue samples. In addition, using proliferating normal vs. human tumor breast cells as a model system, we observed a number of alterations in cyclin expression: (i) an 8-fold amplification of cyclin E gene in one tumor line, a 64-fold overexpression of its mRNA, and altered expression of its protein; (ii) deranged expression of cyclin E protein in all (10 of 10) tumor cell lines studied; (iii) increased cyclin mRNA stability, resulting in (iv) general overexpression of RNAs and proteins for cyclins A and B and CDC2 in 9 of 10 tumor lines and (v) deranged order of appearance of cyclins in synchronized tumor vs. normal cells, with mitotic cyclins appearing prior to G1 cyclins. These multiple general derangements in cyclin expression in human breast cancer cells provide evidence linking aberrant cyclin expression to tumorigenesis.

Breast↗
PubMed1992

Differential display and cloning of messenger RNAs from human breast cancer versus mammary epithelial cells.

Identification of the genes that are specifically expressed in tumor cells but not in normal cells (oncogenes), or vice versa (tumor suppressor genes), is important for understanding the molecular basis of cancer. The differential display technique was applied to compare mRNAs from normal and tumor-derived human mammary epithelial cells, cultured under the same conditions. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. They exhibit characteristics of the 3' end of eukaryotic mRNA, as predicted by the method. A complementary DNA fragment seen only in the normal cell was used as a probe to isolate its corresponding complementary DNA clone from a library. Northern analysis confirmed its differential expression. Thus, this method can be used for detecting, cloning, and sequencing of genes that are unique to a host of biological and disease processes.

Base Sequence↗
PubMed1992

Cloning and characterization of human cyclin D3, a cDNA closely related in sequence to the PRAD1/cyclin D1 proto-oncogene.

Cyclins regulate cell cycle progression by complexing with and activating cdc2 or related kinases. PRAD1/cyclin D1 is a recently discovered putative oncogene in several types of human tumors and may regulate G1-S phase progression. We have cloned a related human cDNA, called cyclin D3, from a placental cDNA library by cross-hybridization with PRAD1. In synchronized HeLa cells, the mRNA levels of PRAD1 and cyclin D3 were regulated reciprocally through the cell cycle: cyclin D3 mRNA levels peaked in S phase, where PRAD1 mRNA was lowest in S. In normal human mammary epithelial (70N) cells synchronized by growth factor deprivation and subsequent growth factor stimulation, PRAD1 expression peaked in G1 and declined before S phase, while cyclin D3 expression rose later in G1 and remained elevated in S. Therefore, the close relationship (53.1% identity) between PRAD1 and cyclin D3 does not necessarily imply redundant functions of these candidate G1 cyclins; they may have distinct roles in progression from G1 through S phase.

Amino Acid Sequence↗
PubMed1992

Down-regulation of a member of the S100 gene family in mammary carcinoma cells and reexpression by azadeoxycytidine treatment.

A cDNA clone, designated CaN19 (originally called clone 19), isolated by subtractive hybridization, contains sequences that are preferentially expressed in normal mammary epithelial cells but not in breast tumor cells. Comparison of its deduced amino acid sequence with sequences in the GenBank data base revealed similarity with the S100 protein family, a group of small Ca(2+)-binding modulator proteins involved in cell cycle progression and cell differentiation. CaN19 expression is down-regulated in normal cells by A23187, a calcium ionophore, suggesting that its regulation is calcium-dependent. We have assigned CaN19 to human chromosome 1q21-q24, a region containing four other S100-related genes. In contrast to CaN19 mRNA expression, most members of the S100 protein family are activated or overexpressed in tumor cells. Synchronization experiments by growth-factor deprivation demonstrated a biphasic induction of CaN19 expression in normal cells, approximately 2-fold in early G1 phase and another 2- to 3-fold at the G1/S boundary. Exposure of mammary tumor cells to 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation, reactivated the expression of CaN19 mRNA.

Amino Acid Sequence↗
PubMed1992

Modification of cell proliferation with inhibitors.

Recent developments on mechanisms that control cell multiplication, using molecular biology, are renewing interest in inhibitors and activators. A great deal of information has been gained in the past through the use of chemicals that modify passage through the cell cycle. The kinds of inhibitors, their sites of action that disrupt functions essential for proliferation, their usefulness in synchronizing cultures and, importantly, their therapeutic value, have been the subject of many investigations.

Animals↗
PubMed1992

Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumor cell lines.

Subtractive hybridization, selecting for mRNAs expressed in normal human mammary epithelial cells (NMECs) but not in mammary tumor cell lines (TMECs), led to the cloning of the human gap junction gene connexin 26 (Cx26), identified by its sequence similarity to the rat gene. Two Cx26 transcripts derived from a single gene are expressed in NMECs but neither is expressed in a series of TMECs. Northern analysis using rat Cx probes showed that Cx43 mRNA is also expressed in the normal cells, but not in the tumor lines examined. Connexin genes Cx31.1, Cx32, Cx33, Cx37, and Cx40 are not expressed in either normal cells or the tumor lines examined. In cell-cell communication studies, the normal cells transferred Lucifer yellow, while tumor cells failed to show dye transfer. Both Cx26 and Cx43 proteins were immunolocalized to membrane sites in normal cells but were not found in tumor cells. Further analysis demonstrated that Cx26 is a cell-cycle regulated gene expressed at a moderate level during G1 and S, and strongly up-regulated in late S and G2, as shown with lovastatin-synchronized NMECs. Cx43, on the contrary is constitutively expressed at a uniform low level throughout the cell cycle. Treatment of normal and tumor cells with a series of drugs: 5dB-cAMP, retinoic acid, okadaic acid, estradiol, or TGFb had no connexin-inducing effect in tumor cells. However, PMA induced re-expression of the two Cx26 transcripts but not of Cx43 in several TMECs. Thus Cx26 and Cx43 are both downregulated in tumor cells but respond differentially to some signals. Modulation of gap-junctional activity by drug therapy may have useful clinical applications in cancer.

Amino Acid Sequence↗
PubMed1991

Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin.

Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in all types of cells, through several cycles and with minimal overall metabolic perturbations, has not yet been available. We describe a procedure for synchronizing normal as well as tumor cells reversibly in the G1 phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This method of synchronization was successful with all cell lines tested, including normal and tumor cells of mouse, hamster, and human origins. For example, when MCF-7 human breast cancer cells were synchronized with Lovastatin and released by the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase), 3 phases of accelerated thymidine incorporation into DNA corresponding to 3 S phases of the cell cycle occurred during a 90-h period of cell replication. Thymidine incorporation was decreased to less than or equal to 4% during the initial lag of 18 h before the first S phase, and maximum incorporation was then achieved after only 6 h. The antibody Ki-67, which detects a nuclear antigen associated with proliferation, was present in cells arrested with Lovastatin. This fact, together with the lack of thymidine incorporation during the initial lag time, indicates that the cells were arrested in the G1 and not in the G0 phase of the cell cycle. Furthermore, in synchronized tumor-derived human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant synthesis of DNA and histone H4 RNA expression could be observed for 2 cycles. Minimal perturbations of general metabolic functions occurred since the rate of RNA, protein, and initial DNA synthesis were unaffected by Lovastatin, as evidenced by [3H]uridine, [3H]leucine, and initial [3H]thymidine incorporation. Finally, while the Lovastatin-induced synchronization was overcome by mevalonic acid, addition of squalene or cholesterol-ethanol had no such effect. Thus, Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G1 phase.

Animals↗