Therapeutic intervention and signaling.

Significant advances in our understanding of intracellular signal transduction pathways have emerged within the past several years. It is now apparent that, under certain circumstances, particular isoforms of Ras can be prenylated by geranylgeranyl protein transferase as well as farnesyl protein transferase. New pathways controlling growth factor-dependent inhibition of apoptosis involving phosphoinositide 3'-hydroxykinase and the protein kinase Akt have also been clarified.

A peptide-doxorubicin 'prodrug' activated by prostate-specific antigen selectively kills prostate tumor cells positive for prostate-specific antigen in vivo.

We covalently linked doxorubicin with a peptide that is hydrolyzable by prostate-specific antigen. In the presence of prostate tumor cells secreting prostate-specific antigen, the peptide moiety of this conjugate, L-377,202, was hydrolyzed, resulting in the release of leucine-doxorubicin and doxorubicin, which are both very cytotoxic to cancer cells. However, L-377,202 was much less cytotoxic than conventional doxorubicin to cells in culture that do not secrete prostate-specific antigen. L-377,202 was approximately 15 times more effective than was conventional doxorubicin at inhibiting the growth of human prostate cancer tumors in nude mice when both drugs were used at their maximally tolerated doses. Nude mice inoculated with human prostate tumor cells secreting prostate-specific antigen showed considerable reductions in tumor burden with minimal total body weight loss when treated with L-377, 202. This improvement in therapeutic index correlated with the selective localization of leucine-doxorubicin and free doxorubicin in tissues secreting prostate-specific antigen after exposure to L-377,202.

Friend murine leukemia virus-induced leukemia is associated with the formation of mink cell focus-inducing viruses and is blocked in mice expressing endogenous mink cell focus-inducing xenotropic viral envelope genes.

In these studies, we have shown data that are consistent with the hypothesis that mink cell focus-inducing viruses (MCF) play an important role in the generation of an erythroproliferative disease developing after injection of certain strains of newborn mice with ecotropic Friend murine leukemia virus (F-MuLV). Resistance to this disease is correlated with the endogenous expression of an MCF/xenotropic virus-gp70-related protein that may interfere with the replication or spread of MCF viruses. These ideas are supported by the following observations: (a) after infection with F-MuLV, only 6/13 strains of mice-developed disease, and studies with crosses between susceptible and resistant strains indicated that resistance was dominant. Although F-MuLV was shown to replicate equally well in all strains tested, viruses coding for MCF-specific viral envelope proteins could be detected only in the spleens of mice from strains that were resistant to F-MuLV-induced disease and not in the spleens of mice from strains that were resistant to F-MuLV-induced disease; (b) a Friend MCF (Fr-MCF) virus isolated from the spleen of an F-MuLV-infected mouse from a susceptible strain induced the same erythroproliferative disease when injected as an appropriate pseudotype into mice from susceptible but not resistant strains of mice; and (c) resistant but not susceptible strains of mice endogenously express MCF/xenotropic virus-related envelope glycoproteins that may be responsible for resistance by blocking receptors for MCF viruses. These results not only indicate that Fr-MCF virus is a crucial intermediate in the induction of disease by F-MuLV, but also suggest that a novel gene, either an MCF/xenotropic virus-related envelope gene or a gene controlling its expression, is responsible for resistance to erythroleukemia induced by F-MuLV.

Class II histocompatibility antigen expression in human melanocytes transformed by Harvey murine sarcoma virus (Ha-MSV) and Kirsten MSV retroviruses.

Human melanocytes infected with Ki-MSV or Ha-MSV, but not amphotropic MuLV, undergo a series of transformation-related changes that are characteristic of malignant melanoma. These are (a) expression of Ia antigens, in particular DP, DQ, and DR class II histocompatibility gene products, (b) a transformed morphology and ability to grow in soft agar, and (c) a 5-10-fold increase in the cell surface expression of GD3 ganglioside. However, other characteristics of melanoma, such as independence from specific growth factors and loss of adenosine deaminase binding protein were not observed. We conclude that viral ras oncogenes initiate early transformation events in melanocytes, and that Ia antigen expression is a transformation marker in this system.

Signaling pathways in apoptosis as potential targets for cancer therapy.

Genetic instability contributes to the origin of cancer as well as to the ability of cancer cells to become resistant to various therapies. Because of this, cytotoxic rather than cytostatic therapies might be most effective against this disease. Many oncogenes and tumor suppressors mediate their effects by interfering with or inducing apoptotic signaling. Thus, apoptotic pathways might be significantly altered in cancer cells relative to untransformed cells, and these differences might present a therapeutic window that can be exploited for development of cancer drugs.

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.

Drug-targeting strategies in cancer therapy.

Genetic changes in cell-cycle, apoptotic, and survival pathways cause tumorigenesis, leading to significant phenotypic changes in transformed cells. These changes in the tumor environment - elevated expression of surface proteases, increased angiogenesis and glucuronidase activity - can be taken advantage of to improve the therapeutic index of existing cancer therapies. Targeting cytotoxics to tumor cells by enzymatic activation is a promising strategy for improving chemotherapeutics.

Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F.

E2F is a mammalian transcription factor that appears to play an important role in cell cycle regulation. While at least two proteins (E2F-1 and DP-1) with E2F-like activity have been cloned, studies from several laboratories suggest that additional homologs may exist. A novel protein with E2F-like properties, designated E2F-2, was cloned by screening a HeLa cDNA library with a DNA probe derived from the DNA binding domain of E2F-1 (K. Helin, J. A. Lees, M. Vidal, N. Dyson, E. Harlow, and A. Fattaey, Cell 70:337-350, 1992). E2F-2 exhibits overall 46% amino acid identity to E2F-1. Both the sequence and the function of the DNA and retinoblastoma gene product binding domains of E2F-1 are conserved in E2F-2. The DNA binding activity of E2F-2 is dramatically enhanced by complementation with particular sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified components of HeLa cell E2F, and anti-E2F-2 antibodies cross-react with components of purified HeLa cell E2F. These observations are consistent with a model in which E2F binds DNA as a heterodimer of two distinct proteins, and E2F-2 is functionally and immunologically related to one of these proteins.

Lovastatin selectively inhibits ras activation of the 12-O-tetradecanoylphorbol-13-acetate response element in mammalian cells.

To evaluate ras-mediated signal transduction, an alkaline phosphatase gene (SEAP) was placed under the control of the ras-inducible phorbol ester response element (TRE) in murine fibroblasts (TRE-SEAP cells). The Kirsten ras gene was placed under the control of the glucocorticoid-inducible mouse mammary tumor virus promoter and introduced into the TRE-SEAP cells. Dexamethasone increased ras expression in the TRE-SEAP cells carrying the Kirsten ras gene and stimulated SEAP activity 25-fold. Lavostatin blocked dexamethasone induction of SEAP activity (50% inhibitory concentration, 0.5 microM) but did not affect phorbol ester-induced SEAP activity in the same cells. Lovastatin also did not block forskolin induction of SEAP activity in cells expressing SEAP under the control of the cyclic AMP response element.

Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction.

Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.

Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein.

Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRB's ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation. These results suggest that pRB60 associates with E7 and E2F through overlapping but distinct domains.

Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha.

Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.

Epidermal growth factor receptor binding is affected by structural determinants in the toxin domain of transforming growth factor-alpha-Pseudomonas exotoxin fusion proteins.

TGF-alpha-PE40 is a hybrid protein composed of transforming growth factor-alpha (TGF-alpha) fused to a 40,000-dalton segment of Pseudomonas exotoxin A (PE40). This hybrid protein possesses the receptor-binding activity of TGF-alpha and the cell-killing properties of PE40. These properties enable TGF-alpha-PE40 to bind to and kill tumor cells that possess epidermal growth factor (EGF) receptors. Unexpectedly, TGF-alpha-PE40 binds approximately 100-fold less effectively to EGF receptors than does native TGF-alpha (receptor-binding inhibition IC50 = 540 and 5.5 nM, respectively). To understand the factors governing receptor binding, deletions and site-specific substitutions were introduced into the PE40 domain of TGF-alpha-PE40. Removal of the N-terminal 59 or 130 amino acids from the PE40 domain of TGF-alpha-PE40 improved receptor binding (IC50 = 340 and 180 nM, respectively) but decreased cell-killing activity. Substitution of alanines for cysteines at positions 265 and 287 within the PE40 domain dramatically improved receptor binding (IC50 = 37 nM) but also decreased cell-killing activity. Similar substitutions of alanines for cysteines at positions 372 and 379 within the PE40 domain did not significantly affect receptor-binding or cell-killing activities. These studies indicate that the PE40 domain of TGF-alpha-PE40 interferes with EGF receptor binding. The cysteine residues at positions 265 and 287 of PE40 are responsible for a major part of this interference.

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.

A farnesyltransferase inhibitor induces tumor regression in transgenic mice harboring multiple oncogenic mutations by mediating alterations in both cell cycle control and apoptosis.

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV-v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV-v-Ha-ras mice were interbred with p53(-/-) mice. Tumors in ras/p53(-/-) mice treated with L-744,832 regressed as efficiently as MMTV-v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV-v-Ha-ras mice were also interbred with MMTV-c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV-v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV-c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.

Tumor necrosis factor not detectable in patients with clinical cancer cachexia.

Tumor necrosis factor (TNF) has been implicated in the pathogenesis of cachexia in neoplastic and infectious diseases. To assess the relationship between TNF and weight loss among cancer patients, we assayed TNF levels in serum from 19 patients who had lost 8%-40% of premorbid weight. The weight loss experienced by these patients was not attributable to anticancer therapy, gastrointestinal disorders, or other medical problems. TNF was measured using a quantitative sandwich enzyme-linked immunosorbent assay that is sensitive to human TNF in serum at concentrations greater than or equal to 40 pg/mL. No TNF was detected in serum samples from the 19 patients studied.

High-density functional gastrin releasing peptide receptors on primate cells.

Gastrin releasing peptide (GRP) is a 27 amino acid hormone that elicits a variety of biological effects. Receptor-binding antagonists of GRP may have therapeutic use in several pathologic conditions including cancer. The identification and characterization of GRP receptor antagonists have been aided by the use of murine 3T3 cells that possess functional GRP receptors. However, no human or primate cell lines that possess high-density GRP receptors and exhibit a biochemical or biological response to GRP have been described. To address this problem, we examined a series of cell lines and found that GRP specifically binds to Cos-7 monkey cells and stimulates elevation of intracellular calcium in these cells. Cos-7 cells exhibit a single class of high-affinity (dissociation constant = 0.13 nM) GRP binding sites (35,000/cell). Cross-linking experiments that use radiolabeled GRP identified two species of putative GRP receptor proteins (relative molecular mass, 90,000 and 22,000). Competitive binding inhibition studies indicate that Cos-7 cells tightly bind GRP-specific receptor antagonists. These antagonists block the binding of radiolabeled GRP to Cos-7 cells and inhibit GRP-stimulated elevation of intracellular calcium. These properties make Cos-7 cells a useful reagent for the study of GRP receptor antagonists.

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.

Protective influence of lactoferrin on mice infected with the polycythemia-inducing strain of Friend virus complex.

Purified iron-saturated human lactoferrin (LF) was assessed in vivo for effects on the survival rates of C57BL X DBA/2 f1 (hereafter called BD2F1) (Fv-2sr) mice and titers of spleen focus-forming viruses (SFFV) in BD2F1 and DBA/2 (Fv-2ss) mice inoculated with the polycythemia-inducing strain of the Friend virus complex (FVC-P). LF prolonged the survival rates and decreased the titers of SFFV in mice given FVC-P. Titers of SFFV, assayed 14 days after administration of FVC-P, were measured by the spleen focus-forming unit assay in secondary mouse recipients. Decreases in titers of SFFV were apparent when LF was given in vivo as a single bolus dose of 200 micrograms within 2 h of the Friend virus complex (FVC), or as a total dosage of 200 micrograms given on days 1, 2, 4, 7, 9, and 11 after FVC-P, and to a lesser degree when LF was given as a total dosage of 200 micrograms on days 3, 4, 7, 9, and 11 after FVC-P. No decreases in titers of SFFV were detected when LF was given up to 3 days before or more than 3 days after FVC-P. LF did not appear to be directly inactivating the viruses as it did not inactivate the SFFV or the Friend murine leukemia helper virus in vitro. The results suggest that the protective effect of LF in vivo is probably due to an action on cells responding to the FVC or to an action on cells which influence the cells responding to the FVC or which influence the virus. It has been shown elsewhere that LF decreases the percentage of marrow and spleen hematopoietic progenitor cells that are in DNA synthesis in vivo and this may be the means by which the protective effect of LF is mediated in mice given the FVC.