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A Oliff

Publications and source records attributed to A Oliff.

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

Cloning and mutagenesis of the p110 alpha subunit of human phosphoinositide 3'-hydroxykinase.

Activation of phosphoinositide 3'-hydroxykinase (P13K) is required for mitogenic signal transduction by several growth factors and oncogenes. P13K is a heterodimer consisting of a p85 regulatory subunit and a p110 catalytic subunit. In the current study, we report the cloning and characterization of the p110 alpha catalytic subunit of human P13K. This clone is highly homologous (> 99% amino acid identity) to bovine brain p110 alpha, but contains 10 amino acid differences from the human p110 alpha sequence previously reported. Comparison of this sequence with known Ser/Thr kinases and p110 homologs highlighted several conserved residues within the putative kinase domain. Mutational analysis of these residues (Asp915, (Asp933 + Phe934)) yielded P13K mutants with virtually complete loss of phosphoinositide phosphorylating activity. Expression of the wild-type p110 alpha protein in CHO cells is sufficient to activate the serum response element derived from the promoter of c-fos, an immediate early gene product. In contrast, the catalytically impaired p110 alpha mutants as well as the p85 alpha subunit of P13K were inactive in the fos assay. These studies suggest that the mitogenic signal transduction pathway mediated by P13K is dependent upon the enzymatic activity of the p110 alpha subunit of P13K.

Amino Acid Sequence↗
PubMed1997

Farnesyltransferase inhibitors versus Ras inhibitors.

Over the past few years, the idea that farnesyl-protein transferase (FPTase) inhibitors might be effective antiproliferative/antitumor agents has been realized in studies of cultured cells and in rodent models of cancer. Most of the studies with FPTase inhibitors have focused on inhibiting the growth of ras-transformed cells in vitro or the growth of ras-dependent tumors in mice. More recently, it has been recognized that the antiproliferative effect of FPTase inhibitors may extend beyond ras-driven tumors. It now seems likely that the ability of FPTase inhibitors to reverse the malignant phenotype results, at least in part, from inhibiting the farnesylation of proteins other than Ras.

Alkyl and Aryl Transferases↗
PubMed1997

The potential of farnesyltransferase inhibitors as cancer chemotherapeutics.

Mutant ras oncogenes and alterations in the mitogenic signaling pathways that they regulate are associated with a wide variety of solid tumors and leukemias for which existing chemotherapeutics have limited utility. Of the possible approaches to inhibit Ras function, much attention has focused on a posttranslational modification, farnesylation, which is required for the subcellular localization of Ras to the plasma membrane and is critical to Ras cell-transforming activity. Inhibitors of the enzyme that catalyzes Ras farnesylation, farnesyl-protein transferase (FPTase), have been developed. These compounds inhibit the tumorigenic phenotypes of ras-transformed cells and human tumor cells in cell culture and in animal models. Moreover, FPTase inhibitors have not demonstrated toxicity to normal cells in culture or to normal tissues in mice. FPTase inhibitors are among the first small molecule compounds designed from studies of oncogenes that might serve as novel cancer chemotherapeutics.

Alkyl and Aryl Transferases↗
PubMed1996

Selection of potent inhibitors of farnesyl-protein transferase from a synthetic tetrapeptide combinatorial library.

Inhibitors of farnesyl-protein transferase (FPTase) show promise as anticancer agents. Based on the sequence of the protein substrates of FPTase (the CAAX sequence), potent and selective peptidomimetic inhibitors have been developed; these compounds share with the peptide substrate a free thiol and a C-terminal carboxylate. We have used a synthetic tetrapeptide combinatorial library to screen for new leads devoid of these features: the peptides were C-terminally amidated, and no free thiol was included in the combinatorial building blocks. To compensate for this negative bias, an expanded set of 68 amino acids was used, including both L and D as well as many non-coded residues. Sixteen individual tetrapeptides derived from the consensus were synthesized and tested; all were active, showing IC50 values ranging from low micromolar to low nanomolar. The most active peptide, D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid (Ki = 2 nM), is also very selective showing little inhibitory activity against the related enzyme geranylgeranyl-protein transferase type I (IC50 > 50 microM). In contrast to CAAX-based peptidomimetics, D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma-carboxyglut amic acid appeared to mimic the isoprenoid substrate farnesyl diphosphate as determined by kinetic and physical measurements. D-Tryptophan-Dmethionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid was a competitive inhibitor of FPTase with respect to farnesyl diphosphate substrate and uncompetitive with respect to CAAX substrate. Furthermore, we demonstrated that FPTase undergoes ligand dependent conformational changes in its circular dichroism spectrum and that D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid induced a conformational change identical to that observed with farnesyl diphosphate ligand.

Alkyl and Aryl Transferases↗
PubMed1996

Farnesyltransferase inhibitors and anti-Ras therapy.

The oncoprotein encoded by mutant ras genes is initially synthesized as a cytoplasmic precursor which requires posttranslational processing to attain biological activity; farnesylation of the cysteine residue present in the CaaX motif located at the carboxy-terminus of all Ras proteins is the critical modification. Once farnesylated and further modified, the mature Ras protein is inserted into the cell's plasma membrane where it participates in the signal transduction pathways that control cell growth and differentiation. The farnesylation reaction that modifies Ras and other cellular proteins having an appropriate CaaX motif is catalyzed by a housekeeping enzyme termed farnesyl-protein transferase (FPTase). Inhibitors of this enzyme have been prepared by several laboratories in an effort to identify compounds that would block Ras-induced cell transformation and thereby function as Ras-specific anticancer agents. A variety of natural products and synthetic organic compounds were found to block farnesylation of Ras proteins in vitro. Some of these compounds exhibit antiproliferative activity in cell culture, block the morphological alterations associated with Ras-transformation, and can block the growth of Ras-transformed cell lines in tumor colony-forming assays. By contrast, these compounds do not affect the growth or morphology of cells transformed by the Raf or Mos oncoproteins, which do not require farnesylation to achieve biological activity. The efficacy and lack of toxicity observed with FPTase inhibitors in an animal tumor model suggest that specific FPTase inhibitors may be useful for the treatment of some types of cancer.

Alkyl and Aryl Transferases↗
PubMed1995

A peptidomimetic inhibitor of farnesyl:protein transferase blocks the anchorage-dependent and -independent growth of human tumor cell lines.

Farnesyl protein transferase (FPTase) catalyzes the first of a series of posttranslational modifications of Ras required for full biological activity. Peptidomimetic inhibitors of FPTase have been designed that selectively block farnesylation in vivo and in vitro. These inhibitors prevent Ras processing and membrane localization and are effective in reversing the transformed phenotype of Rat1-v-ras cells but not that of cells transformed by v-raf or v-mos. We have tested the effect of the FPTase inhibitor L-744,832 (FTI) on the anchorage-dependent and -independent growth of human tumor cell lines. The growth of over 70% of all tumor cell lines tested was inhibited by 2-20 microM of the FTI, whereas the anchorage-dependent growth of nontransformed epithelial cells was less sensitive to the effects of the compound. No correlation was observed between response to drug and the origin of the tumor cell or whether it contained mutationally activated ras. In fact, cell lines with wild-type ras and active protein tyrosine kinases in which the transformed phenotype may depend on upstream activation of the ras pathway were especially sensitive to the drug. To define the important targets of FTI action, the mechanism of cellular drug resistance was examined. It was not a function of altered drug accumulation or of FPTase insensitivity since, in all cell lines tested, FPTase activity was readily inhibited within 1 h of treatment with the inhibitor. Furthermore, the general pattern of inhibition of cellular protein farnesylation and the specific inhibition of lamin B processing were the same in sensitive and resistant cells. In addition, functional activation of Ras was inhibited to the same degree in sensitive and resistant cell lines. However, the FTI inhibited the epidermal growth factor-induced activation of mitogen-activated protein kinases in sensitive cells but not in two resistant cell lines. These data suggest that the drug does inhibit ras function and that resistance in some cells is associated with the presence of Ras-independent pathways for mitogen-activated protein kinase activation by tyrosine kinases. We conclude that FPTase inhibitors are potent antitumor agents with activity against many types of human cancer cell lines, including those with wild-type ras.

Alkyl and Aryl Transferases↗
PubMed1995

An SH3 domain is required for the mitogenic activity of microinjected phospholipase C-gamma 1.

Phospholipase activity is elevated in dividing cells. In response to growth factor stimulation, phospholipase C-gamma (PLC-gamma) binds to activated tyrosine kinase receptors via SH2 binding domains, resulting in phosphorylation of PLC-gamma and activation of its enzyme activity. These observations suggest that PLC-gamma participates in the signal transduction pathway employed by growth factors to promote mitogenesis. Consistent with this hypothesis, microinjection of purified bovine PLC-gamma into quiescent fibroblasts has been previously reported to initiate a mitogenic response [Smith et al. (1989) Proc. Natl. Acad. Sci. 86, 3659]. We have reproduced this result using recombinant rat PLC-gamma protein. Surprisingly, however, a catalytically inactive mutant of PLC-gamma, H335Q, also elicited a full mitogenic response. The capacity to induce mitogenesis by microinjection of PLC-gamma was mapped to the 'Z' domain of the protein, which contains PLC-gamma's SH2 and SH3 motifs. Inactivation of the phosphorylated tyrosine binding properties of both SH2 domains had no effect on the mitogenic activity of the Z-domain peptide. However, deletion of the SH3 domain resulted in a complete loss of activity. These results suggest that PLC-gamma's mitogenic properties do not require the enzyme's phospholipase activity, but are instead mediated by a novel pathway for mitogenic stimulation which is dependent upon an intact SH3 domain.

3T3 Cells↗
PubMed1995

Development of inhibitors of protein farnesylation as potential chemotherapeutic agents.

Protein prenylation, adding either the 15-carbon isoprenoid farnesyl or the 20-carbon isoprenoid geranylgeranyl to cysteine residue(s) at or near the C-termini of proteins, is a recently identified post-translational modification that localizes some proteins to a membrane compartment. One of the most intensely studied prenylated proteins is Ras, a low molecular weight GTP-binding protein that plays an important role in the regulation of cell proliferation. Proteins encoded by ras genes with oncogenic mutations are capable of transforming cells in culture. Such mutated ras genes are frequently found in a wide variety of human tumors. Localization of the Ras oncoprotein to the cytoplasmic face of the plasma membrane via farnesylation is essential for efficient cell transforming ability. Thus, inhibition of the Ras farnesylation reaction is a possible anti-cancer strategy. Several strategies have been employed to inhibit Ras farnesylation, including inhibition of isoprenoid biosynthesis and inhibition of the enzyme which catalyzes the farnesylation reaction, farnesyl-protein transferase (FPTase). Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate limiting enzyme in isoprenoid biosynthesis, inhibit Ras farnesylation and block the growth of ras-transformed cells. However, antiproliferative effects do not result from specific inhibition of Ras farnesylation; they are also observed in cells transformed by raf, which is independent of Ras farnesylation. A more specific approach to inhibiting Ras farnesylation is to inhibit FPTase. Using random screening of natural products and a rational design approach, a variety of compounds that specifically inhibit FPTase have been isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Identification of a novel retinoblastoma gene product binding site on human papillomavirus type 16 E7 protein.

Transformation of mammalian cells by human papillomavirus type 16 appears to require binding of the viral E7 protein to the cellular retinoblastoma growth suppressor gene product (pRB). Binding of E7 protein to pRB inhibits several of pRB's biochemical properties, including association with the transcription factor E2F. Fragments of E7 protein derived from its conserved region 2 (CR2) domain bind to pRB and are sufficient to inhibit binding of full-length E7 protein to pRB. However, these CR2 fragments exhibit reduced affinity for pRB compared to the full-length protein and do not inhibit formation of the pRB-E2F complex. These observations suggest the existence of additional contact sites between the E7 protein and pRB. In the current study we have identified a region of E7, distinct from the CR2 domain, which is sufficient to bind pRB. This new pRB binding motif encompasses the zinc-binding conserved region 3 (CR3) domain of E7. Studies with a series of pRB deletion mutants suggest that pRB residues between amino acids 803 and 841 are necessary for binding to the E7 CR3 domain. An E7 CR3 peptide inhibits binding of E2F to pRB, indicating that E2F and E7(31-98) bind to pRB at the same or overlapping sites. These results are consistent with a model in which optimal binding of E7 to pRB requires at least two distinct contact sites: the previously identified high affinity interaction between the E7 CR2 domain and the pRB "pocket" region, and a second interaction between the E7 CR3 domain and the COOH-terminal region of pRB. The latter interaction is sufficient for E7's inhibition of E2F binding to pRB.

Amino Acid Sequence↗
PubMed1994

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.

Actins↗
PubMed1993

Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor.

To acquire transforming potential, the precursor of the Ras oncoprotein must undergo farnesylation of the cysteine residue located in a carboxyl-terminal tetrapeptide. Inhibitors of the enzyme that catalyzes this modification, farnesyl protein transferase (FPTase), have therefore been suggested as anticancer agents for tumors in which Ras contributes to transformation. The tetrapeptide analog L-731,735 is a potent and selective inhibitor of FPTase in vitro. A prodrug of this compound, L-731,734, inhibited Ras processing in cells transformed with v-ras. L-731,734 decreased the ability of v-ras-transformed cells to form colonies in soft agar but had no effect on the efficiency of colony formation of cells transformed by either the v-raf or v-mos oncogenes. The results demonstrate selective inhibition of ras-dependent cell transformation with a synthetic organic inhibitor of FPTase.

Alkyl and Aryl Transferases↗
PubMed1993

Transcription factor E2F binds DNA as a heterodimer.

E2F is a mammalian transcription factor that appears to play an important role in cell cycle control. DNA affinity column-purified E2F from HeLa cells reproducibly exhibits multiple protein bands when analyzed by SDS/PAGE. After electrophoretic purification, electroelution, and refolding of the individual protein components, the E2F DNA binding activity of the individual proteins was poor. However, upon mixing the individual components together, a dramatic (100- to 1000-fold) increase in specific DNA binding activity was observed. The five protein bands isolated can be separated into two groups based on apparent molecular mass. Optimal reconstitution of activity requires one of the two proteins found in the group of larger molecular mass (approximately 60 kDa) and one of the three proteins in the smaller-sized group (approximately 50 kDa). The reconstituted heterodimer is identical to authentic affinity-purified E2F by three criteria: DNA-binding specificity, DNA pattern, and binding to the retinoblastoma gene product. A recently cloned protein with E2F-like activity, RBP3/E2F-1, is related to the protein components of the group of larger molecular mass, as determined by Western blot analysis and reconstitution experiments. These data suggest that E2F, like many other transcription factors, binds DNA as an oligomeric complex composed of at least two distinct proteins.

Adenoviruses, Human↗
PubMed1993

Selective inhibition of farnesyl-protein transferase blocks ras processing in vivo.

The ras oncogene product, Ras, is synthesized in vivo as a precursor protein that requires post-translational processing to become biologically active and to be capable of transforming mammalian cells. Farnesylation appears to be a critical modification of Ras, and thus inhibitors of the farnesyl-protein transferase (FPTase) that catalyzes this reaction may block ras-dependent tumorigenesis. Three structural classes of FPTase inhibitors were identified: (alpha-hydroxyfarnesyl)phosphonic acid, chaetomellic acids, and zaragozic acids. By comparison, these compounds were weaker inhibitors of geranylgeranyl-protein transferases. Each of these inhibitors was competitive with respect to farnesyl diphosphate in the FPTase reaction. All compounds were assayed for inhibition of Ras processing in Ha-ras-transformed NIH3T3 fibroblasts. Ras processing was inhibited by 1 microM (alpha-hydroxyfarnesyl)phosphonic acid. Neither chaetomellic acid nor zaragozic acid were active in this assay. These results are the first demonstration that a small organic chemical selected for inhibition of FPTase can inhibit Ras processing in vivo.

3T3 Cells↗