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Importance of the Stress Kinase p38 in Mediating the Direct Cytotoxic Effects of the Thalidomide Analogue, CPS49, in Cancer Cells and Endothelial Cells

Authors' Affiliations: ¹ Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland and ² Clinical Pharmacology Research Core, Science Applications International Corporation-Frederick, Inc., National Cancer Institute-Frederick, Frederick, Maryland

Requests for reprints: Phillip A. Dennis, National Cancer Institute/Navy Medical Oncology, Room 5101, Building 8, 8901 Wisconsin Avenue, Bethesda, MD 20889. Phone: 301-496-0929; Fax: 301-435-4345; E-mail: pdennis{at}nih.gov.

	Abstract

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Purpose: Thalidomide has gained renewed interest as a cancer therapeutic due to its potential antiangiogenic effects. The thalidomide analogues CPS11 and CPS49 are active in preclinical angiogenesis assays and xenograft model systems, but the biochemical basis for these observations is unclear.

Experimental Design: To address this question, we assessed the toxicity of these thalidomide analogues in cancer cells, endothelial cells, and genetically modified cells using assays that measure apoptotic and nonapoptotic cell death. Phosphospecific and native antibodies were used in immunoblotting and immunohistochemical experiments to assess the activation states of kinases that control cellular survival in vitro and in vivo.

Results: CPS49 predominantly induced nonapoptotic cell death in lung cancer cells, prostate cancer cells, and endothelial cells in a dose-dependent manner, whereas CPS11 was not cytotoxic. CPS49 did not inhibit kinases that promote survival, such as Akt or extracellular signal-regulated kinase, but rather rapidly activated the stress kinase p38 pathway in both cancer cells and endothelial cells. CPS49 activated p38 in tumor xenografts. Using p38–/– cells or an inhibitor of p38, we show that the presence and activation of p38 is important for cytotoxicity in all cell types examined.

Conclusions: Our studies identify a unifying mechanism of action for cytotoxicity of the tetraflourinated thalidomide analogue, CPS49, and suggest that activation of p38 could serve as a biomarker in clinical trials with CPS49.

The antiangiogenic effects of thalidomide have been evaluated in several clinical trials involving a variety of cancers. Thalidomide has clinical activity in multiple myeloma (3, 4), hepatocellular carcinoma (5), Kaposi's sarcoma (6), and prostate cancer (7, 8). Thalidomide is believed to require metabolic activation to generate its antiangiogenic effect. Biotransformation of thalidomide by cytochrome P450 2C19 isoform seems to produce the metabolite responsible for the activity of the drug (9). To improve the anticancer activity of thalidomide, analogues were recently synthesized and tested in preclinical models (10). These analogues differ in structure as either N-substituted or tetrafluorinated classes of thalidomide. Both classes of thalidomide analogues significantly inhibit angiogenesis in rat aortic ring assays, as well as decrease human umbilical vascular endothelial cell (HUVEC) proliferation and tube formation (11). The most promising N-substituted analogue (CPS11) and tetrafluorinated analogue (CPS49) were chosen for this study because they were shown to be effective at biologically relevant doses in vitro and in vivo.

Although thalidomide and its analogues possess antiangiogenic properties, a critical mechanism of action has heretofore not been identified. In this report, we show that CPS49 is directly cytotoxic to cancer cells and endothelial cells, and that its cytotoxic effects are dependent on the activation of p38 mitogen-activated protein kinase (MAPK). Moreover, activation of p38 by CPS49 was observed in tumor xenografts. Because activation of the stress kinase p38 is a common feature of cancer therapy, especially chemotherapy and radiation therapy, p38 activation could be an important clinical determinant of response to thalidomide and/or its analogues.

	Materials and Methods

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Thalidomide analogues. The CPS11 and CPS49 thalidomide analogues were synthesized as previously described (11). The drugs were dissolved in DMSO (Sigma-Aldrich, Milwaukee, WI) to a stock concentration of 200 mmol/L and stored at –20°C until use.

Cell culture. H157 cells, PC3 cells, H1703 cells, as well as wild-type (WT) mouse embryonic fibroblasts (MEF) and p38–/– MEF (kind gifts from Dr. Michael Karin, University of California, San Diego) were maintained in 75 cm² flasks in RPMI 1640 or DMEM (Life Technologies, Gaithersburg, MD) with the addition of 10% fetal bovine serum (FBS) plus 1% penicillin-streptomycin (Life Technologies). For growth factor deprivation and treatment experiments, cells were cultured in medium supplemented with 0.1% FBS. HUVEC cells were maintained in 750 cm² flasks in EBM-2 medium supplemented with Endothelial Basal Cell Medium Bullet kit (Cambrex, Westchester, PA). For treatment experiments, cells were cultured in EBM-2 medium without the Bullet kit. All treatments were done with low FBS concentrations or the absence of exogenous growth factors to minimize the contribution of serum components or growth factors on activation of signaling pathways and to isolate the effects of CPS49 or CPS11. All cells were incubated at 37°C in a 5% CO₂ atmosphere.

Viability assays. For measurement of viability, 2 x 10⁵ cells per well were plated in six-well plates. Following overnight recovery in complete medium, cells were treated with 10 µmol/L of the pharmacologic inhibitor SB203580 for 30 minutes in medium containing 0.1% FBS. Control samples received DMSO alone. CPS11 and CPS49 analogues were then added directly to the wells in the appropriate doses. Twenty-four hours after treatment, floating and adherent cells were harvested and isolated by centrifugation for 10 minutes at 1,300 rpm. Cell pellets were stained with trypan blue dye solution. Respective samples of stained cell suspensions were counted in triplicate on a hemocytometer.

Average cell viability was calculated by dividing viable cells by total cell count. For biochemical analysis of the p38 MAPK pathway in response to pharmacologic treatments, 2 x 10⁵ cells per well were plated on six-well plates in parallel with the viability assays and described above. Cells were harvested after 2-hour treatment with thalidomide analogues, and cell lysates were prepared and processed for immunoblot evaluation of the p38 MAPK pathway as described below. Viability assays were done at least thrice.

Apoptosis assays. Cells were treated in triplicate with 50 µmol/L CPS49 or CPS11 for 24 or 48 hours. Floating and adherent cells were harvested and centrifuged at 1,350 rpm for 10 minutes. Cells were fixed in 70% methanol for 30 minutes at –20°C and then stained with 10 µg/mL propidium iodide for 30 minutes at room temperature. Apoptosis was assessed by flow cytometry. Apoptosis experiments were done in triplicate and repeated at least thrice.

Immunoblotting. Cell lysates were prepared and collected by washing cells with PBS, followed by lysis with 2x Laemmli sample buffer containing 20% glycerol, 0.5 mol/L Tris (pH 6.8), 10% SDS, and supplemented with protease inhibitor cocktail (Roche, Branchburg, NJ). Lysates were then sonicated for 20 seconds, and the protein yield was quantitated using the Bio-Rad detergent-compatible protein assay kit. Immunoblot analysis was done as previously described (12). Total protein and phosphospecific antibodies against Akt (S473), extracellular signal-regulated kinase (ERK; T202/Y204), p38 (T180/Y182), HSP27 (S82), and ATF2 (T71) were obtained from Cell Signaling Technology (Beverly, MA). The antibody against -tubulin was obtained from Sigma-Aldrich. Immunoblotting experiments were done thrice.

Animal studies. National Cancer Institute-Frederick is accredited by Association for Assessment and Accreditation of Laboratory Animal Care and follows the USPHS policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the Guide for Care and Use of Laboratory Animals (National Research Council, 1996. National Academy Press, Washington, DC) PC3 cells (3 x 10⁶) were injected s.c. into each flank of 5 to 6-week-old male severe combined immunodeficient mice. Tumors were allowed to grow until palpable (4 weeks). Animals were treated once with i.p. injections of either vehicle (0.5% carboxymethylcellulose) or CPS49 (25 mg/kg). Tumors were harvested at 30 minutes, 2 hours, or 4 hours. Harvested tumors were either homogenized in 2x LSB using a dounce homogenizer and processed for immunoblotting or fixed in 4% paraformaldehyde (PBS, pH 7.4) and processed for immunohistochemistry. After 24 hours, fixed tumors were placed in 80% ethanol until sectioning.

Immunohistochemistry. Sections from each fixed tumor were stained with H&E for histologic examination. For detection of phospho-p38, sections were stained with the immunohistochemically preferred rabbit monoclonal phospho-p38 MAPK antibody (1:50; Cell Signaling Technology). High pH antigen retrieval buffer (Roche) and 3,3'-diaminobenzidine (Sigma-Aldrich) reagents were used. The protocol from the manufacturer was followed and antigens were visualized using the streptavidin-biotin-peroxidase method.

	Results

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CPS49 is cytotoxic to non–small cell lung cancer cells in a dose-dependent manner. To determine whether CPS11 and CPS49 were toxic to non–small cell lung carcinoma cells, H157 cells were incubated for 24 or 48 hours in medium containing DMSO or different doses (20-100 µmol/L) of CPS11 or CPS49. Samples were collected and stained with trypan blue to assess cell viability. At doses as high as 100 µmol/L and an incubation time of 48 hours, CPS11 was not toxic to H157 cells (Fig. 1A ). However, CPS49 induced extensive cell death in H157 cells. At 24 and 48 hours, CPS49 decreased viability in a dose-dependent manner. Treatment with 20 µmol/L CPS49 reduced cell viability to 91%, and treatment with 50 µmol/L reduced viability below 70%. The most dramatic effects were seen with 100 µmol/L treatment, which decreased the number of viable cells to below 8% compared with DMSO control samples (Fig. 1A). These data show that despite structural similarity, CPS11 was not an effective cytotoxic agent, but that CPS49 was able to effectively kill lung cancer cells in a dose-dependent manner.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Dose-dependent cytotoxicity of thalidomide analogues in non–small cell lung carcinoma cells. A, H157 cells were treated with different doses of CPS11 or CPS49 in medium containing 0.1% FBS for 24 or 48 hours. Floating and adherent cells were harvested, stained with trypan blue, and counted using a hemocytometer to assess viability. B, H157 cells were treated with 50 µmol/L CPS11 or CPS49 in medium containing 0.1% FBS for 24 or 48 hours. Cells were then harvested, fixed, and stained with propiduim iodide. Apoptosis was measured using flow cytometry. *, P < 0.05.

CPS49 activates the MAPKs p38 and ERK in non–small cell lung carcinoma cells and prostate cancer cells. To investigate signal transduction mechanisms that might be responsible for the cytotoxic effects of CPS49, we focused primarily on key kinases that control cellular survival. First, we investigated Akt, which is responsible for enhanced cell survival when activated. Levels of phosphorylated Akt were not inhibited by CPS49 or CPS11 in H157 cells (Fig. 2A ). In fact, CPS49 increased Akt activation after 1 hour exposure. Levels of total Akt did not change. Finding no evidence of Akt inhibition, we assessed ERK, a MAPK that promotes cellular survival and cellular proliferation. CPS49 markedly increased ERK phosphorylation within 15 minutes and activation was maintained for 2 hours. In contrast, CPS11 only slightly increased ERK phosphorylation at 2 and 4 hours. Levels of total ERK did not change. We also examined p38, a member of the MAPK superfamily that promotes necrosis and/or apoptosis when activated. CPS49, and to a much lesser extent CPS11, activated p38 within 15 minutes. Activation of p38 by CPS49 peaked at 30 minutes. Activation of p38 was transient because the level of p38 phosphorylation decreased to basal levels by 1 hour. To establish that the CPS49-induced p38 signal was being propagated, phosphorylation of two p38 downstream substrates, HSP27 and ATF2, was analyzed. Phosphorylation of HSP27 and ATF2 increased with similar kinetics as was observed with p38, but only HSP27 phosphorylation decreased in parallel with p38 phosphorylation. Levels of total p38 did not change. Similar effects on Akt, ERK, and p38 after administration of CPS49 or CPS11 were observed with another non–small cell lung carcinoma cell line, H1703 (data not shown). Thus, CPS49 rapidly and transiently activates two MAPKs in H157 cells, ERK and p38, but does not inhibit Akt activation.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Effects of thalidomide analogues on activation of Akt, ERK, and p38. A, H157 cells were treated with CPS11 or CPS49 (20 µmol/L) for the times indicated and lysates were prepared for immunoblotting. Activation of Akt, ERK, p38, HSP27, and ATF2 were assessed using phosphospecific antibodies. Total levels of Akt, ERK, and p38 protein were included to show that total protein is not affected by CPS treatment. -Tubulin was used as a loading control. B, experiments were done as in (A), except that PC3 cells were used. C, H157, PC3, and HUVEC cells were treated with the indicated concentrations of CPS49 in medium containing 0.1% FBS. Two hours after treatment, lysates were collected and prepared for immunoblotting. Activation of HSP27 and p38 was assessed using phosphospecific antibodies. Total p38 was used as a loading control.

CPS49 activates the p38 pathway in a dose-dependent manner. To correlate the dose-dependent cytotoxicity of CPS49 with p38 activation, we examined the response of the p38 pathway to increasing doses of CPS49 (Fig. 2C). In two cancer cell lines (H157 and PC3) and human umbilical vein endothelial cells (HUVEC), CPS49 increased phosphorylation of p38 and HSP27 in a dose-dependent manner. The most dramatic increases in p38 activation were observed between 10 and 200 µmol/L, which parallels the dose-dependent decrease in viability in H157 cells (Fig. 1A). At 20 µmol/L CPS49, levels of phosphorylated p38 and HSP27 remain unchanged in the cancer cell lines but are considerably increased in HUVEC. This enhanced sensitivity of HUVEC to p38 pathway activation by CPS49 is consistent with greater sensitivity of HUVEC to the inhibitory effects of CPS49 on cellular proliferation that has been observed previously (11). Together, these data show that concentrations of CPS49 that induce cellular changes also induce activation of the p38 pathway. Moreover, they suggest that the magnitude of p38 activation determines cytotoxicity.

A p38 inhibitor decreases CPS49-induced cytotoxicity in cancer cells. To establish whether activation of ERK or p38 by CPS49 was meaningful in cancer cells, we tested whether pharmacologic inhibitors of MAP/ERK kinase (U0126), the kinase upstream of ERK, or p38 (SB203580) could affect the response to CPS49. Consistent with earlier studies from our group, U0126 effectively inhibited ERK phosphorylation in H157 cells (13), but did not affect CPS49-induced cytotoxicity (data not shown). In contrast, inhibition of p38 with SB203580 reduced the cytotoxic effects of CPS49 in H157 cells (Fig. 3 ). H157 cells treated for 24 hours with 100 µmol/L CPS49 alone were only 8% viable, but pretreatment with SB203580 increased viability after exposure to 100 µmol/L CPS49 to 76%. Increased viability caused by SB203580 correlated with inhibition of p38 and HSP27 phosphorylation. Similar results were observed with PC3 cells (data not shown). At 100 µmol/L CPS, only 32% of PC3 cells were viable at 24 hours. If PC3 cells were pretreated with SB203580 and CPS49 was administered under the same conditions, 65% of the cells were viable. Increased viability of PC3 cells with SB203580 pretreatment was again associated with decreased phosphorylation of p38 and HSP27. These data indicate that CPS-induced cytotoxicity in cancer cell lines correlates with p38 activation but not ERK activation.

View larger version (15K): [in this window] [in a new window]   Fig. 3. CPS49 cytotoxicity in cancer cells is dependent on the activation of p38 MAPK. H157 cells were pretreated with SB203580 (10 µmol/L) for 30 minutes or medium alone. CPS49 was then added at the doses indicated for 24 hours. Control samples were treated with DMSO or SB alone. Samples were stained with trypan blue to assess viability. Parallel samples of were collected for immunoblotting (insets). *, P < 0.05.

View larger version (25K): [in this window] [in a new window]   Fig. 4. CPS49 activates the p38 MAPK signaling pathway in endothelial cells and is cytotoxic in a p38-dependent manner. A, HUVEC cells were treated with CPS49 (20 µmol/L) for the indicated times. Activation of Akt, p38, HSP27, and ATF2 were assessed using phosphospecific antibodies. Total levels of Akt, ERK, and p38 protein were included to show that total protein is not affected by CPS treatment. -Tubulin was used as a loading control. B, p38-dependent cytotoxicity in HUVEC cells was assessed using SB203580 as described in Fig. 3. *, P < 0.05.

Effect of CPS49 in WT and p38–/– MEFs. To further establish that p38 activation promotes the cytotoxic effects of CPS49, we used genetically modified MEFs that do not express p38 (p38–/– MEFs), as well as MEFs derived from WT littermates (WT MEFs). Viability assays with each cell type were done after exposure to different doses of CPS11 and CPS49 for 24 or 48 hours (Fig. 5A and B , respectively). Similar to our results in cancer cell lines, CPS11 had no toxic effect on either the WT MEFs or the p38–/– MEFs at doses as high as 100 µmol/L. In contrast, CPS49 was extremely toxic to WT MEFs, decreasing viability in a dose-dependent manner at 24 and 48 hours. Although CPS49 was toxic to the p38–/– MEFs at the highest dose tested (100 µmol/L), p38–/– MEFs were much more resistant to CPS49 than WT MEFs. For example, after 48 hours treatment with 20 µmol/L CPS49, p38–/– MEFs were over 98% viable, compared with only 18% viability in WT MEFs that express p38. The fact that p38–/– MEFs died in response to very high doses of CPS49 perhaps reflects their different species of origin, their fibroblastic nature, or their immortalization with viral proteins. It may also indicate that other isoforms of p38, such as p38ß, p38, or p38, that are normally expressed at much lower levels than p38 might be activated by high doses of CPS49, or that at these doses in this cell type, cytotoxic mechanisms other than p38 activation are used. Nonetheless, these studies are consistent with our results in cancer cells and HUVEC and show that activation of p38 is largely responsible for CPS49-mediated cytotoxicity in immortalized fibroblasts.

View larger version (15K): [in this window] [in a new window]   Fig. 5. CPS49-induced cytotoxicity in immortalized fibroblasts is dependent on activation of p38 MAPK. A, p38–/– MEF or WT MEFs were treated with different doses of CPS11 or CPS49 for 24 hours. Floating and adherent cells were then harvested, stained with trypan blue, and counted using a hemocytometer in triplicate to assess viability. B, cells were treated as in (A), except that the incubation with CPS analogues was extended to 48 hours. C, p38-dependent cytotoxicity in WT MEF cells was assessed using SB203580, as described in Fig. 3A. *, P < 0.05.

CPS49 activates p38 in vivo. The development of new cancer therapeutic agents might be expedited if biological markers of drug administration could be identified. To test whether p38 activation might serve as a biomarker for CPS49 activity, we assessed p38 activation in vivo. PC3 cells were grown as xenografts in the flanks of severe combined immunodeficient mice and animals were injected i.p. once with either the drug vehicle or CPS49 (25 mg/kg). Tumors were harvested at 30 minutes, 2 hours, and 4 hours for immunohistochemistry and immunoblot analysis. Immunoblotting of tumor extracts showed that PC3 tumors derived from CPS49-treated animals had increased p38 phosphorylation at all three time points (Fig. 6 ). Total p38 levels did not change. The greatest induction was seen 30 minutes after injection, indicating that CPS49 quickly achieves effective circulating concentrations. Immunohistochemistry was also done on tumor sections from each time point to assess p38 activation. Similar to the data obtained in the tumor lysates, intensity of staining with the phospho-p38 antibody increased in tumors after CPS49 treatment (30-minute time point shown; Fig. 6). Although active p38 was detectable in nuclear and cytoplasmic compartments in the vehicle-treated animals, staining with phospho-p38 increased in both compartments as a result of CPS49 treatment. These experiments show that CPS49 activates p38 in vivo and further suggests that p38 activation could serve as a biomarker for CPS49 administration.

View larger version (34K): [in this window] [in a new window]   Fig. 6. CPS49 activates p38 in vivo. The flanks of severe combined immunodeficient mice were injected s.c. with PC3 cells (3 x 106) and tumors were allowed to develop until palpable (4 weeks). Animals were treated once with i.p. injections of either the drug vehicle (0.5% carboxymethylcellulose) or CPS49 (25 mg/kg). Tumors were harvested at 30, 120, and 240 minutes after treatment and were prepared for immunoblotting with phosphospecific and total p38 antibodies. Tumors were also prepared for immunohistochemistry. Slides from tumors harvested after 30 minutes were stained with phospho-p38 antibodies. Control slides were processed after omitting the primary antibody.

	Discussion

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Despite the structural similarities of these analogues, CPS49, but not CPS11, proved to be toxic to lung cancer cells and prostate cancer cells in a dose-dependent manner. Activation of p38, but not inhibition of Akt or ERK, correlated with cytotoxicity. Instead of decreased phosphorylation, increased phosphorylation of Akt and ERK was observed in lung cancer and prostate cancer cells in response to CPS49. This paradoxical, transient increase in Akt activation has been observed after administration of many types of chemotherapy, as well as administration of CPS49 or CPS11 (15, 16). Likewise, Kumar et al. (16) observed prolonged induction of ERK phosphorylation in myeloma cells after administration of CPS49 and CPS11. It is possible that activation of ERK and Akt are compensatory responses in cancer cells to cellular stress induced by p38 activation. In such a case, cancer cells might activate these survival signals to combat the cytotoxic stimulus produced by p38 activation. Such a reciprocal relationship between p38 and Akt has been observed previously (17). In addition, Kumar et al. (16) showed that a MAP/ERK kinase inhibitor increased the cytotoxicity of thalidomide analogues in myeloma cells, supporting the notion that ERK activation is a compensatory response that promotes cellular survival.

Interestingly, the Akt and ERK signaling pathways responded differently to CPS49 and CPS11 in endothelial cells. HUVEC showed decreased activation of Akt and a lack of induction of ERK in response to both analogues. Thus, the cumulative effects on p38, Akt, and ERK could explain the sensitivity of endothelial cells to thalidomide analogues. In addition to differences in p38, Akt, and ERK, the status of other pathways such as signal transducers and activators of transcription 3 (STAT3) could contribute to the cell type specificity of thalidomide analogues. For example, Kumar et al. (16) showed that CPS11 inhibits STAT3 phosphorylation and induces apoptosis of myeloma cells. Activation of p38 by CPS11 was not assessed before 2 hours in this study, so the role of p38 activation in CPS-induced apoptosis of myeloma cells is unclear. It is possible that transient activation of p38 could have occurred and could have contributed to the cellular response of myeloma cells to CPS11. The relative increased activation of Akt and ERK by cancer cells versus HUVEC suggests that cancer cells might have a more effective method of producing compensatory survival signals to stave off cell death.

Using biochemical and genetic methods of inhibition, we were able to establish that CPS49 has profound toxic effects that directly correlate with p38 activation in a diverse array of cell lines. p38 is an important member of the MAPK superfamily and is activated in response to various cell stresses such as changes in osmolarity, DNA damage, heat shock, ionizing radiation, inflammatory cytokines, and ischemia (18). Activation of the p38 pathway can induce a number of cellular responses including necrosis and apoptosis. Therefore, activation of p38 might be desirable in cancer cells or tumor vascular cells, but would be undesirable in normal cells because it might cause toxicity. In fact, many autoimmune and inflammatory disorders are characterized by p38 activation (19).

There are several hypothetical implications of p38 induction when considering the development of thalidomide analogues as cancer drugs. First, p38 activation is important for the response of cancer cells to several chemotherapeutic agents (20–23), which shows that chemotherapy and CPS49 share induction of the same stress kinase pathway that mediates killing of cancer cells, and suggests that thalidomide analogues could be considered as legitimate, direct cytotoxic cancer agents. Second, CPS49-mediated p38 activation could contribute to an antiangiogenic effect not only because of direct effects on endothelial cells, but also because inhibition of p38 has been shown to enhance vascular endothelial growth factor–induced angiogenesis in vitro and in vivo (24). Third, activation of p38 by CPS49 could be used as a biomarker to facilitate pharmacodynamic analysis. Phosphospecific antibodies against p38 could be used in immunohistochemical analysis of tissues to measure induction of p38 activation in response thalidomide analogues in vivo (25). In support of this, we were able perform immunoblotting and immunohistochemistry with commercially available phosphospecific and total p38 antibodies to show that CPS49 activates p38 in vivo. The identification of p38 activation as a unifying mechanism of action provides an explanation for the cytotoxic effects and antiangiogenic effects of thalidomide analogues, and could be exploited to facilitate their continued clinical development.

	Footnotes

 
Grant support: Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, and federal funds from the National Cancer Institute, NIH, under contract N01-CO-12400 (E.R. Lepper).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: This research is conducted in partial fulfillment for The Johns Hopkins University/National Cancer Institute Center for Cancer Research degree in Molecular Targets and Drug Discovery Technologies. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Received 8/21/05; revised 3/13/06; accepted 3/23/06.

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