Two peptides derived from ras-p21 induce either phenotypic reversion or tumor cell necrosis of ras-transformed human cancer cells.

PURPOSE: We investigated the effects of two peptides from the ras-p21 protein, corresponding to residues 35-47 (PNC-7) and 96-110 (PNC-2), on two ras-transformed human cancer cell lines, HT1080 fibrosarcoma and MIAPaCa-2 pancreatic cancer cell lines. In prior studies, we found that both peptides block oncogenic, but not insulin-activated wild-type, ras-p21-induced oocyte maturation. When linked to a transporter penetratin peptide, these peptides induce reversion of ras-transformed rat pancreatic cancer cells (TUC-3) to the untransformed phenotype. METHODS: These peptides and a control peptide, linked to a penetratin peptide, were incubated with each cell lines. Cell counts were obtained over several weeks. The cause of cell death was determined by measuring caspase as an indicator of apoptosis and lactate dehydrogenase (LDH) as marker of necrosis. Since both peptides block the phosphorylation of jun-N-terminal kinase (JNK) in oocytes, we blotted cell lysates of the two cancer cell lines for the levels of phosphorylated JNK to determine if the peptides reduced these levels. RESULTS: We find that both peptides, but not control peptides linked to the penetratin sequence, induce phenotypic reversion of the HT-1080 cell line but cause tumor cell necrosis of the MIA-PaCa-2 cell line. On the other hand, neither peptide has any effect on the viability of an untransformed pancreatic acinar cell line, BMRPA1. We find that, while total JNK levels remain constant during peptide treatment, phosphorylated JNK levels decrease dramatically, consistent with the mechanisms of action of these peptides. CONCLUSION: We conclude that these peptides block tumor but not normal cell growth likely by blocking oncogenic ras-p21-induced phosphorylation of JNK, an essential step on the oncogenic ras-p21-protein pathway. These peptides are therefore promising as possible anti-tumor agents.

Site-specific phosphorylation of raf in cells containing oncogenic ras-p21 is likely mediated by jun-N-terminal kinase.

In a study of interactions between the raf-MEK-MAPK (ERK) and JNK-jun pathways, we found previously that JNK can induce phosphorylation of raf but not vice versa. In this study, we investigate the nature of the JNK-induced phosphorylation of raf. In in vitro experiments in which immunobead-bound raf is phosphorylated by activated JNK, we find strong phosphorylation signals at raf-Ser259 and Ser338. The Ser259 phosphorylation is surprising since it is associated with inhibition of migration of raf to the cell membrane where it can interact with ras-p21. We also find that in oocytes induced to mature with oncogenic ras-p21, which induces high levels of phosphorylated JNK and MAPK, the same pattern of phosphorylation of raf occurs. In contrast, in oocytes induced to mature with insulin, which requires activation of wild-type ras-p21, phosphorylation of raf-Ser338 but not raf-Ser259 occurs. In oncogenic ras-transformed human pancreatic cancer MIA-PaCa-2 cells, phosphorylation of both raf serines occurs. Treatment of these cells with the ras peptide, PNC-2 attached to a penetrating sequence that blocks JNK and MAPK phosphorylation and induces tumor cell necrosis, results in a marked decrease in phosphorylation of raf-Ser259, but not that of raf-Ser338. These results suggest that oncogenic ras-p21 induces phosphorylation of both raf-Ser259 and Ser338 and that raf-Ser 259 phosphorylation may be effected by activated JNK.

The dual-specificity kinases, TOPK and DYRK1A, are critical for oocyte maturation induced by wild-type--but not by oncogenic--ras-p21 protein.

We have previously found that oncogenic ras-p21 and insulin, which activates wild-type ras-21 protein, both induce Xenopus laevis oocyte maturation that is dependent on activation of raf. However, oncogenic ras-p21 utilizes raf-dependent activation of the two classic raf targets, MEK and MAP kinase (MAPK or ERK) while insulin-activated wild-type ras-p21 does not depend on activation of these two kinases. Utilizing a microarray containing the entire Xenopus genome, we discovered two dual specificity kinases, T-Cell Origin Protein Kinase (TOPK), known to bind to raf and the nuclear kinase, DYRK1A, that are expressed at much higher levels in insulin-matured oocytes. Using SiRNA's directed against expression of both of these proteins, we now show that each inhibits insulin-but not oncogenic ras-p21-induced oocyte maturation. Control siRNA's have no effect on either agent in induction of maturation. We find that each SiRNA "knocks down" expression of its target protein while not affecting expression of the other protein. These results suggest that both proteins are required for maturation induced by wild-type, but not oncogenic, ras-p21. They also suggest that oncogenic and wild-type ras-p21 utilize pathways that become divergent downstream of raf. On the basis of these findings, we propose a model for two signal transduction pathways by oncogenic and activated wild-type ras-p21 showing points of overlap and divergence.

Rapid conformational dynamics of cytochrome P450 2E1 in a natural biological membrane environment.

Among the members of the cytochrome P450 superfamily, P450 2E1 is most often associated with the production of reactive oxygen species and subsequent cellular toxicity. We sought to identify a structural basis for this distinguishing feature of P450 2E1 by examining its carbon monoxide binding kinetics as a probe of conformation/dynamics. We employed liver microsomes from wild-type and P450 2E1 knockout mice in order to characterize this P450 in a natural membrane environment. The CO binding kinetics of the P450s of wild-type microsomes had a rapid component that was absent in the knockout microsomes. Data analysis using the maximum entropy method (MEM) correspondingly identified two distinct kinetic components in the wild-type microsomes and only one component in the knockout microsomes. The rapid kinetic component in wild-type microsomes was attributed to endogenous P450 2E1, while the slower component was derived from the remaining P450s. In addition, rapid binding kinetics and a single component were also observed for human P450 2E1 in a baculovirus expression system, in the absence of other P450s. Binding kinetics of both mouse and human P450 2E1 were slowed in the presence of ethanol, a modulator of this P450. The unusually rapid CO binding kinetics of P450 2E1 indicate that it is more dynamically mobile than other P450s and thus able to more readily interconvert among alternate conformations. This suggests that conformational switching during the catalytic cycle may promote substrate release from a short-lived binding site, allowing activated oxygen to attack other targets with toxic consequences.

Preferential induction of necrosis in human breast cancer cells by a p53 peptide derived from the MDM2 binding site.

p53 is the most frequently altered gene in human cancer and therefore represents an ideal target for cancer therapy. Several amino terminal p53-derived synthetic peptides were tested for their antiproliferative effects on breast cancer cell lines MDA-MB-468 (mutant p53), MCF-7 (overexpressed wild-type p53), and MDA-MB-157 (null p53). p53(15)Ant peptide representing the majority of the mouse double minute clone 2 binding site on p53 (amino acids 12-26) fused to the Drosophila carrier protein Antennapedia was the most effective. p53(15)Ant peptide induced rapid, nonapoptotic cell death resembling necrosis in all breast cancer cells; however, minimal cytotoxicity was observed in the nonmalignant breast epithelial cells MCF-10-2A and MCF-10F. Bioinformatic/biophysical analysis utilizing hydrophobic moment and secondary structure predictions as well as circular dichroism spectroscopy revealed an alpha-helical hydrophobic peptide structure with membrane disruptive potential. Based on these findings, p53(15)Ant peptide may be a novel peptide cancer therapeutic because it induces necrotic cell death and not apoptosis, which is uncommon in traditional cancer therapy.

Molecular modeling of mammalian cytochrome P450s.

The cytochrome P450 enzymes collectively metabolize a wide range of xenobiotic and endogenous compounds. The broad substrate specificity of this superfamily derives from the multiplicity of P450s whose unique substrate specificity profiles reflect underlying differences in primary sequence. Experimental structures of P450s, where available, have provided great insight into the basis of substrate recognition. However, for those mammalian P450s whose structures have not been determined, homology modeling has become an increasingly important tool for understanding substrate specificity and mechanism. P450 modeling is often a challenging task, owing to the rather low sequence identity between target and template proteins. Although mammalian P450 models have previously been based on bacterial P450 structures, the recent advent of mammalian P450 structures holds great potential for generating more accurate homology models. Consequently, the substrate recognition properties of several mammalian P450s have been rationalized using the predicted substrate binding site of recently developed models. This review summarizes the major concepts and current approaches of molecular modeling of P450s.

Arginine to lysine 108 substitution in recombinant CYP1A2 abolishes methoxyresorufin metabolism in lymphoblastoid cells.

1. Cytochrome P4501A2 (CYP1A2) activates a large number of procarcinogens to carcinogens. Phytochemicals such as flavones can inhibit CYP1A2 activity competitively, and hydroxylated derivatives of flavone (galangin) may be potent, selective inhibitors of CYP1A2 activity relative to CYP1A1 activity. Molecular modelling of the CYP1A2 interaction with hydroxylated derivatives of flavone suggests that a number of hydrophobic residues of the substrate-binding domain engage in hydrogen bonding with such inhibitors. 2. We have tested this model using site-directed mutagenesis of these residues in expression plasmids transfected into the human B-lymphoblastoid cell line, AHH-1 TK+/-. 3. Consistent with the molecular model's predicted placement in the active site, amino acid substitutions at the predicted residues abolished CYP1A2 enzymatic activity. 4. Transfected cell lines contained equal amounts of immunoreactive CYP1A2. 5. Our results support the molecular model's prediction of the critical amino acid residues present in the hydrophobic active site, residues that can hydrogen bond with CYP1A2 inhibitors and modify substrate binding and/or turnover.

Peptides designed from molecular modeling studies of the ras-p21 protein induce phenotypic reversion of a pancreatic carcinoma cell line but have no effect on normal pancreatic acinar cell growth.

PURPOSE: From molecular modeling studies we found that two ras-p21 peptides, corresponding to p21 residues 35-47 (PNC-7) and 96-110 (PNC-2), selectively block oncogenic (Val 12-p21), but not insulin-activated wild-type, p21-induced oocyte maturation. Our purpose was to determine if these peptides block the growth of mammalian cancer cells but not their normal counterpart cells. METHODS: Since oncogenic ras has been implicated as a causative factor in over 90% of human pancreatic cancers, we have established a normal pancreatic acinar cell line (BMRPA1) and the corresponding ras-transformed pancreatic cancer cell line (TUC-3). We treated both cell lines with PNC-7 and PNC-2 and the unrelated negative control peptide, X13, attached to the penetratin sequence that allows membrane penetration and also transfected these cell lines with plasmids encoding all three peptides. RESULTS: Treatment of TUC-3 cells with each peptide resulted in their complete phenotypic reversion to the untransformed phenotype as revealed by the lack of tumor formation of these revertant cells implanted in the peritoneal cavities of nude mice. In contrast, treatment with X13-leader resulted in no inhibition of cell growth. Identical results were obtained when TUC-3 cells were transfected with plasmids expressing PNC-2, PNC-7 and X13. None of these peptides affected the normal growth of BMRPA1 cells. CONCLUSIONS: PNC-2 and PNC-7 peptides induce phenotypic reversion of ras-induced pancreatic cancer cells and have no effect on normal pancreatic cell growth. Since the plasmid encoding PNC-2 without penetratin also had the same effect on the TUC-3 cell line, we conclude that the penetratin sequence has no effect on the activity of this peptide. Since X13 attached to penetratin had no effect on TUC-3 cells, the effect is specific for PNC-2 and PNC-7 and further confirms that the effect is not caused by the penetratin sequence. PNC-2- and PNC-7-penetratin may therefore be useful in the treatment of ras-induced pancreatic carcinomas.

Comparison of molecular dynamics averaged structures for complexes of normal and oncogenic ras-p21 with SOS nucleotide exchange protein, containing computed conformations for three crystallographically undefined domains, suggests a potential role of these domains in ras signaling.

ras-p21 protein binds to the son-of-sevenless (SOS) guanine nucleotide-exchange promoter that allows it to exchange GDP for GTP. Previously, we performed molecular dynamics calculations on oncogenic (Val 12-) and wild-type ras-p21 bound to SOS. By superimposing the average structures of these two complexes, we identified four domains (residues 631-641, 676-691, 718-729, and 994-1004) in SOS that change conformation and were candidates for being effector domains. These calculations were performed in the absence of three crystallographically undefined loops (i.e., residues 591-596, 654-675, and 742-751). We have now modeled these loops into the SOS structure and have re-performed the dynamics calculations. We find that all three loop domains undergo large changes in conformation that involve mostly changes in their positioning and not their individual conformations. We have also identified another potential effector domain (i.e., residues 980-989). Overall, our current results suggest that SOS interactions with oncogenic ras-p21 may enhance ras-p21 mitogenic signaling through prolonging its activation by maintaining its binding to GTP and by allowing its effector domains to interact with intracellular targets.

An effector peptide from glutathione-S-transferase-pi strongly and selectively blocks mitotic signaling by oncogenic ras-p21.

Oncogenic ras-p21 directly activates jun-N-terminal kinase (JNK) and its substrate, jun as a unique step on its mitogenic signal transduction pathway. This activation is blocked by the specific JNK-jun inhibitor, glutathione-S-transferase-pi (GST-pi). Four domains of GST-pi have been implicated in this regulatory function: 34-50, 99-121, 165-182, and 194-201. The 34-50 domain is unique in that it does not affect GST-pi binding to JNK-jun but blocks jun phosphorylation by JNK. We now find that it completely blocks oncogenic (Val 12-) ras-p21-induced oocyte maturation but has no effect on insulin-induced oocyte maturation. Because the latter process requires activation of wild-type ras-p21, this peptide appears to be specific for inhibiting only the oncogenic form of ras-p21, suggesting its use in treating ras-induced tumors.

Loop domain peptides from the SOS ras-guanine nucleotide exchange protein, identified from molecular dynamics calculations, strongly inhibit ras signaling.

In the accompanying paper, we found, using molecular dynamics calculations, four domains of the ras-specific SOS guanine nucleotide exchange protein (residues 589-601, 654-675, 746-761, and 980-989) that differ markedly in conformation when SOS is complexed with either oncogenic (Val 12-) ras-p21 or wild-type ras-p21. Three of these domains contain three crystallographically undefined loops that we modeled in these calculations, and one is a newly identified non-loop domain containing SOS residues 980-989. We have now synthesized peptides corresponding to these four domains and find that all of them block Val 12-ras-p21-induced oocyte maturation. All of them also block insulin-induced oocyte maturation, but two of these peptides, corresponding to SOS residues 589-601 and 980-989, block oncogenic ras to a significantly greater extent. These results suggest that SOS contains domains, including the three loop domains, that are important for ras signaling and that several of these domains can activate different pathways specific to oncogenic or wild-type ras-p21.

Identification of 2-piperidone as a biomarker of CYP2E1 activity through metabolomic phenotyping.

Cytochrome P450 2E1 (CYP2E1) is a key enzyme in the metabolic activation of many low molecular weight toxicants and also an important contributor to oxidative stress. A noninvasive method to monitor CYP2E1 activity in vivo would be of great value for studying the role of CYP2E1 in chemical-induced toxicities and stress-related diseases. In this study, a mass spectrometry-based metabolomic approach was used to identify a metabolite biomarker of CYP2E1 through comparing the urine metabolomes of wild-type (WT), Cyp2e1-null, and CYP2E1-humanized mice. Metabolomic analysis with multivariate models of urine metabolites revealed a clear separation of Cyp2e1-null mice from WT and CYP2E1-humanized mice in the multivariate models of urine metabolomes. Subsequently, 2-piperidone was identified as a urinary metabolite that inversely correlated to the CYP2E1 activity in the three mouse lines. Backcrossing of WT and Cyp2e1-null mice, together with targeted analysis of 2-piperidone in mouse serum, confirmed the genotype dependency of 2-piperidone. The accumulation of 2-piperidone in the Cyp2e1-null mice was mainly caused by the changes in the biosynthesis and degradation of 2-piperidone because compared with the WT mice, the conversion of cadaverine to 2-piperidone was higher, whereas the metabolism of 2-piperidone to 6-hydroxy-2-piperidone was lower in the Cyp2e1-null mice. Overall, untargeted metabolomic analysis identified a correlation between 2-piperidone concentrations in urine and the expression and activity of CYP2E1, thus providing a noninvasive metabolite biomarker that can be potentially used in to monitor CYP2E1 activity.

PNC-28, a p53-derived peptide that is cytotoxic to cancer cells, blocks pancreatic cancer cell growth in vivo.

PNC-28 is a p53 peptide from its mdm-2-binding domain (residues 17-26), which contains the penetratin sequence enabling cell penetration on its carboxyl terminal end. We have found that this peptide induces necrosis, but not apoptosis, of a variety of human tumor cell lines, including several with homozygous deletion of p53, and a ras-transformed rat acinar pancreatic carcinoma cell line, BMRPA1. Tuc3. On the other hand, PNC-28 has no effect on untransformed cells, such as rat pancreatic acinar cells, BMRPA1, and human breast epithelial cells and no effect on the differentiation of human stem cells. In this study, we now test PNC-28 in vivo for its ability to block the growth of BMRPA1. Tuc3 cells. When administered over a 2-week period in the peritoneal cavities of nude mice containing simultaneously transplanted tumors, PNC-28 causes complete destruction of these tumors. When delivered concurrently with tumor explantation at a remote site, PNC-28 causes a complete blockade of any tumor growth during its 2-week period of administration and 2 weeks posttreatment, followed by weak tumor growth that plateaus at low tumor sizes compared with tumor growth in the presence of a control peptide. When administered after tumor growth has occurred at a site remote from the tumor, PNC-28 causes a decrease in tumor size followed by a slow increase in tumor growth that is significantly slower than growth in the presence of control peptide. These results suggest that PNC-28 may be effective in treating cancers especially if delivered directly to the tumor.

Functional interactions of Raf and MEK with Jun-N-terminal kinase (JNK) result in a positive feedback loop on the oncogenic Ras signaling pathway.

In previous studies we have found that oncogenic (Val 12)-ras-p21 induces Xenopus laevis oocyte maturation that is selectively blocked by two ras-p21 peptides, 35-47, also called PNC-7, that blocks its interaction with raf, and 96-110, also called PNC-2, that blocks its interaction with jun-N-terminal kinase (JNK). Each peptide blocks activation of both JNK and MAP kinase (MAPK or ERK) suggesting interaction between the raf-MEK-ERK and JNK-jun pathways. We further found that dominant negative raf blocks JNK induction of oocyte maturation, again suggesting cross-talk between pathways. In this study, we have undertaken to determine where these points of cross-talk occur. First, we have immunoprecipitated injected Val 12-Ha-ras-p21 from oocytes and found that a complex forms between ras-p21 raf, MEK, MAPK, and JNK. Co-injection of either peptide, but not a control peptide, causes diminished binding of ras-p21, raf, and JNK. Thus, one site of interaction is cooperative binding of Val 12-ras-p21 to raf and JNK. Second, we have injected JNK, c-raf, and MEK into oocytes alone and in the presence of raf and MEK inhibitors and found that JNK activation is independent of the raf-MEK-MAPK pathway but that activated JNK activates raf, allowing for activation of ERK. Furthermore, we have found that constitutively activated MEK activates JNK. We have corroborated these findings in studies with isolated protein components from a human astrocyte (U-251) cell line; that is, JNK phosphorylates raf but not the reverse; MEK phosphorylates JNK but not the reverse. We further have found that JNK does not phosphorylate MAPK and that MAPK does not phosphorylate JNK. The stress-inducing agent, anisomycin, causes activation of JNK, raf, MEK, and ERK in this cell line; activation of JNK is not inhibitable by the MEK inhibitor, U0126, while activation of raf, MEK, and ERK are blocked by this agent. These results suggest that activated JNK can, in turn, activate not only jun but also raf that, in turn, activates MEK that can then cross-activate JNK in a positive feedback loop.

Comparison of the average structures, from molecular dynamics, of complexes of GTPase activating protein (GAP) with oncogenic and wild-type ras-p21: identification of potential effector domains.

GTPase activating protein (GAP) is a known regulator of ras-p21 activity and is a likely target of ras-induced mitogenic signaling. The domains of GAP that may be involved in this signaling are unknown. In order to infer which domains of GAP may be involved, we have performed molecular dynamics calculations of GAP complexed to wild-type and oncogenic (Val 12-containing) ras-p21, both bound to GTP. We have computed and superimposed the average structures for both complexes and find that there are four domains of GAP that undergo major changes in conformation: residues 821-851, 917-924, 943-953, and 1003-1020. With the exception of the 943-953 domain, none of these domains is involved in making contacts with ras-p21, and all of them occur on the surface of the protein, making them good candidates for effector domains. In addition, three ras-p21 domains undergo major structural changes in the oncogenic p21-GAP complex: 71-76 from the switch 2 domain; 100-108, which interacts with SOS, jun and jun kinase (JNK); and residues 122-138. The change in conformation of the 71-76 domain appears to be induced by changes in conformation in the switch 1 domain (residues 32-40) and in the adjacent domain involving residues 21-31. In an accompanying paper, we present results from microinjection of peptides corresponding to each of these domains into oocytes induced to undergo maturation by oncogenic ras-p21 and by insulin-activated wild-type cellular p21 to determine whether these domain peptides may be involved in ras signaling through GAP.

Inhibition of ras-induced oocyte maturation by peptides from ras-p21 and GTPase activating protein (GAP) identified as being effector domains from molecular dynamics calculations.

In the accompanying article, using molecular dynamics calculations, we found that the 66-77 and 122-138 domains in ras-p21 and the 821-827, 832-845, 917-924, 943-953, and 1003-1020 domains in GAP have different conformations in complexes of GAP with wild-type and oncogenic ras-p21. We have now synthesized peptides corresponding to each of these domains and coinjected them into oocytes with oncogenic p21, which induces oocyte maturation, or injected them into oocytes incubated with insulin that induces maturation by activating wild-type cellular ras-p21. We find that all of these peptides inhibit both agents but do not inhibit progesterone-induced maturation that occurs by a ras-independent pathway. The p21 66-77 and 122-138 peptides cause greater inhibition of oncogenic p21. On the other hand, the GAP 832-845 and 1003-1021 peptides inhibit insulin-induced maturation to a significantly greater extent. Since we have found that activated wild-type and oncogenic p21 activate downstream targets like raf differently, these GAP peptides may be useful probes for identifying elements unique to the wild-type ras-p21 pathway.

Identification of the site of inhibition of mitogenic signaling by oncogenic ras-p21 by a ras effector peptide.

We have previously found that a ras switch 1 domain peptide (PNC-7, residues 35-47) selectively blocks oocyte maturation induced by oncogenic (Val 12-containing) ras-p21 protein and also blocks c-raf-induced oocyte maturation. We now find that oncogenic ras-p21 does not inhibit oocyte maturation of a constitutively activated raf protein (raf BXB) that is lacking most of the first 301 amino terminal amino acids, including the major ras binding domain and accessory ras-binding regions. We also find that a dominant negative raf that completely blocks c-raf-induced maturation likewise does not block raf-BXB-induced maturation. We conclude that PNC-7 blocks ras by binding to the amino-terminal domain of raf and that raf BXB must initiate signal transduction in the cytosol.