Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway.

Large granular lymphocyte (LGL) leukemia results from chronic expansion of cytotoxic T cells or natural killer (NK) cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified platelet-derived growth factor (PDGF) as a key master switch in controlling these survival pathways in T-cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGLs of both T- and NK-cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGLs was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-beta transcripts than purified normal CD8(+) T cells or NK cells. We observed that phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream protein kinase B (PKB)/AKT pathways were constitutively activated in both T- and NK-LGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGLs. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long-term survival of leukemic LGLs, may be an important therapeutic strategy.

The pro-apoptotic protein Prostate Apoptosis Response Protein-4 (Par-4) can be activated in colon cancer cells by treatment with Src inhibitor and 5-FU.

The overexpression of the pro-apoptotic protein Prostate Apoptosis Response Protein-4 in colon cancer has been shown to increase response to the chemotherapeutic agent 5-fluorouracil (5-FU). Although colon cancer cells endogenously express Par-4, the presence or overexpression of Par-4 alone does not cause apoptosis. We hypothesize that Par-4 is inactivated in colon cancer. In colon cancer, the levels and the kinase activity of the nonreceptor tyrosine kinase c-Src increase with tumor progression. One of the downstream effectors of c-Src is Akt1. Akt1 has been shown to inhibit the pro-apoptotic activity of Par-4 in prostate cancer cells. We therefore investigated the potential of activating Par-4 by inhibiting c-Src. Colon carcinoma cell lines were treated with the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) in combination with the chemotherapeutic agent 5-FU. Treating cells with PP2 and 5-FU resulted in reduced interaction of Par-4 with Akt1 and with the scaffolding protein 14-3-3σ, and mobilization of Par-4 to the nucleus. Par-4 was shown to interact not only with Akt1 and 14-3-3σ, but also with c-Src. Overexpression of c-Src induced the phosphorylation of Par-4 at tyrosine site/s. Thus, in this study, we have shown that Par-4 can be activated by inhibiting Src with a pharmacological inhibitor and adding a chemotherapeutic agent. The activation of the pro-apoptotic protein Par-4 as reported in this study is a novel mechanism by which apoptosis occurs with a Src kinase inhibitor and 5-FU. In addition, we have demonstrated that the pro-apoptotic activity of endogenously expressed Par-4 can be increased in colon cancer cells.

Synthesis and biological evaluation of a novel class of isatin analogs as dual inhibitors of tubulin polymerization and Akt pathway.

A novel series of 5,7-dibromoisatin analogs were synthesized and evaluated for their cytotoxicities against four human cancer cell lines including colon HT29, breast MCF-7, lung A549 and melanoma UACC903. Analogs 6, 11 and 13 displayed good in vitro anticancer activity on the HT29 human colon cancer cell line in the 1 µM range. Analogs 5, 9 and 12, containing a selenocyanate group in the alkyl chain were the most promising compounds on the breast cancer MCF-7 cell line. Biological assays relating to apoptosis were performed to understand the mechanism of action of these analogs. Compounds 5 and 6 were found to inhibit tubulin polymerization to the same extent as the anticancer drug vinblastine sulfate, but compounds 11 and 13 inhibited significantly better than vinblastine. Further western blot analysis suggested that compound 6 at 2 µM reduced both levels and phosphorylation state of Akt. Compounds 11 and 13 at 1 µM caused reduced Akt protein levels and strongly suppressed the phosphorylation of Akt. Therefore, 11 and 13 were demonstrated as efficient dual inhibitors of both tubulin polymerization and the Akt pathway and good candidates for further study. More importantly, the strategy of microtubule and Akt dual inhibitors might be a promising direction for developing novel drugs for cancer.

Prostate apoptosis response protein 4 sensitizes human colon cancer cells to chemotherapeutic 5-FU through mediation of an NF kappaB and microRNA network.

BACKGROUND: Diminished expression or activity of prostate apoptosis response protein 4 (Par-4) has been demonstrated in a number of cancers, although reports on Par-4 expression during colon cancer progression are lacking. An understanding of the molecular events in conjunction with the genetic networks affected by Par-4 is warranted. RESULTS: Colon cancer specimens derived from patients have significantly diminished expression of Par-4 mRNA relative to paired normal colon. Hence, the functional consequences of reintroducing Par-4 into HT29 colon cancer cells were assessed. Overexpression augmented the interaction of Par-4 with NF kappaB in the cytosol but not nucleus, and facilitated apoptosis in the presence of 5-fluorouracil (5-FU). Analogous findings were obtained when AKT1 pro-survival signaling was inhibited. Transcriptome profiling identified approximately 700 genes differentially regulated by Par-4 overexpression in HT29 cells. Nearly all Par-4-regulated genes were shown by promoter analysis to contain cis-binding sequences for NF kappaB, and meta-analysis of patient expression data revealed that one-third of these genes exist as a recurrent co-regulated network in colon cancer specimens. Sets of genes involved in programmed cell death, cell cycle regulation and interestingly the microRNA pathway were found overrepresented in the network. Noteworthy, Par-4 overexpression decreased NF kappaB occupancy at the promoter of one particular network gene DROSHA, encoding a microRNA processing enzyme. The resulting down-regulation of DROSHA was associated with expression changes in a cohort of microRNAs. Many of these microRNAs are predicted to target mRNAs encoding proteins with apoptosis-related functions. Western and functional analyses were employed to validate several predictions. For instance, miR-34a up-regulation corresponded with a down-regulation of BCL2 protein. Treating Par-4-overexpressing HT29 cells with a miR-34a antagomir functionally reversed both BCL2 down-regulation and apoptosis by 5-FU. Conversely, bypassing Par-4 overexpression by direct knockdown of DROSHA expression in native HT29 cells increased miR-34a expression and 5-FU sensitivity. CONCLUSION: Our findings suggest that the initiation of apoptotic sensitivity in colon cancer cells can be mediated by Par-4 binding to NF kappaB in the cytoplasm with consequential changes in the expression of microRNA pathway components.

Molecular profiling of LGL leukemia reveals role of sphingolipid signaling in survival of cytotoxic lymphocytes.

T-cell large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of CD3(+)CD8(+) cells. Leukemic LGLs correspond to terminally differentiated effector-memory cytotoxic T lymphocytes (CTLs) that escape Fas-mediated activation-induced cell death (AICD) in vivo. The gene expression signature of peripheral blood mononuclear cells from 30 LGL leukemia patients showed profound dysregulation of expression of apoptotic genes and suggested uncoupling of activation and apoptotic pathways as a mechanism for failure of AICD in leukemic LGLs. Pathway-based microarray analysis indicated that balance of proapoptotic and antiapoptotic sphingolipid-mediated signaling was deregulated in leukemic LGLs. We further investigated sphingolipid pathways and found that acid ceramidase was constitutively overexpressed in leukemic LGLs and that its inhibition induced apoptosis of leukemic LGLs. We also showed that S1P(5) is the predominant S1P receptor in leukemic LGLs, whereas S1P(1) is down-regulated. FTY720, a functional antagonist of S1P-mediated signaling, induced apoptosis in leukemic LGLs and also sensitized leukemic LGLs to Fas-mediated death. Collectively, these results show a role for sphingolipid-mediated signaling as a mechanism for long-term survival of CTLs. Therapeutic targeting of this pathway, such as use of FTY720, may have efficacy in LGL leukemia.

Tumor necrosis factor alpha induces p53 up-regulated modulator of apoptosis expression in colorectal cancer cell lines.

PURPOSE: Inflammatory bowel disease (IBD)-associated colorectal carcinogenesis involves dysregulation of multiple cellular pathways, including p53 signaling and cytokine action. The purpose of the current study was to evaluate the effects of tumor necrosis factor alpha (TNF-alpha) on p53 and p53 up-regulated modulator of apoptosis (PUMA), a downstream effector of p53 in the apoptotic pathway in colorectal cancer cells. METHODS: The cell lines HT29 (which express mutant p53) and HCT116 (which express wild-type p53) were treated with TNF-alpha (0, 50, 100, or 500 ng/mL) for 1, 12, 24, or 48 hours. Protein expression and subcellular localization of p53 and PUMA were determined by immunoblot and immunofluorescence. Changes in p53 and PUMA mRNA expression were determined by quantitative real time polymerase chain reaction. RESULTS: Nuclear p53 expression was increased in TNF-alpha-treated HT29 cells; in contrast, expression was decreased or minimally changed in TNF-alpha-treated HCT116 cells, as determined by immunoblot and immunofluorescence. At 24 hours, p53 mRNA transcript levels were minimally increased in HT29 cells, whereas PUMA increased 34-fold. CONCLUSIONS: TNF-alpha increased nuclear p53 expression in HT29 cells, which express p53 mutation, but not in HCT116 cells, which are wild type for p53. In addition, TNF-alpha markedly up-regulated PUMA mRNA levels in HT29 cells. Our findings suggest that TNF-alpha may be a factor in carcinogenesis in IBD in cells carrying a p53 mutation.

Src kinase induces tumor formation in the c-SRC C57BL/6 mouse.

Src kinase has been linked as a causative agent in the progression of a number of cancers including colon, breast, lung and melanoma. Src protein and activity levels are increased in colorectal cancer and liver metastases arising secondary to colon cancer. However, although Src protein is increased in colon cancer as early as the adenomatous polyp stage, a role for Src in carcinogenesis has not been established. We developed the c-SRC transgenic mouse in the C57BL/6 strain to address the issue of carcinogenesis in cells with high levels of Src expression. The transgene was constructed with the human c-SRC gene downstream of the mouse metallothionein promoter to create zinc inducible gene expression. In these C57BL/6 mice, Src protein was increased in a number of tissues both with and without zinc induction. No additional carcinogenic agent was administered. After 20 months, mice were assessed for tumor development in the liver and GI tract, as well as other organs. Of the mice with the transgene, 15% developed tumors in the liver while no tumors were detected in wild type C57BL/6 mice. A further study was conducted by crossing c-SRC C57BL/6 mice with p21 nullizygous mice to determine the effect of oncogene expression combined with inactivation of the tumor suppressor gene, p21. Addition of the c-SRC transgene to the p21-/- background increased tumor formation almost 3-fold, while it increased metastasis 6-fold. The data from our study show, for the first time, that Src kinase may play a role in carcinogenesis.

Role of Stat3 in regulating p53 expression and function.

Loss of p53 function by mutation is common in cancer. However, most natural p53 mutations occur at a late stage in tumor development, and many clinically detectable cancers have reduced p53 expression but no p53 mutations. It remains to be fully determined what mechanisms disable p53 during malignant initiation and in cancers without mutations that directly affect p53. We show here that oncogenic signaling pathways inhibit the p53 gene transcription rate through a mechanism involving Stat3, which binds to the p53 promoter in vitro and in vivo. Site-specific mutation of a Stat3 DNA-binding site in the p53 promoter partially abrogates Stat3-induced inhibition. Stat3 activity also influences p53 response genes and affects UV-induced cell growth arrest in normal cells. Furthermore, blocking Stat3 in cancer cells up-regulates expression of p53, leading to p53-mediated tumor cell apoptosis. As a point of convergence for many oncogenic signaling pathways, Stat3 is constitutively activated at high frequency in a wide diversity of cancers and is a promising molecular target for cancer therapy. Thus, repression of p53 expression by Stat3 is likely to have an important role in development of tumors, and targeting Stat3 represents a novel therapeutic approach for p53 reactivation in many cancers lacking p53 mutations.

Iterative microarray and RNA interference-based interrogation of the SRC-induced invasive phenotype.

Src kinase has long been recognized as a factor in the progression of colorectal cancer and seems to play a specific role in the development of the metastatic phenotype. In spite of numerous studies conducted to elucidate the exact role of Src in cancer progression, downstream targets of Src remain poorly understood. Gene expression profiling has permitted the identification of large sets of genes that may be functionally interrelated but it is often unclear as to which molecular pathways they belong. Here we have developed an iterative approach to experimentally reconstruct a network of gene activity regulated by Src and contributing to the invasive phenotype. Our strategy uses a combination of phenotypic anchoring of gene expression profiles and loss-of-function screening by way of RNA-mediated interference. Using a panel of human colon cancer cell lines exhibiting differential Src-specific activity and invasivity, we identify the first two levels of gene transcription responsible for the invasive phenotype, where first-tier genes are controlled by Src activity and the second-tier genes are under the influence of the first tier. Specifically, perturbation of first-tier gene activity by either pharmacologic inhibition of Src or RNA-mediated interference-directed knockdown leads to a loss of invasivity and decline of second-tier gene activity. The targeting of first-tier genes may be bypassed altogether because knockdown of second-tier genes led to a similar loss of invasive potential. In this manner, numerous members of a "transcriptional cascade" pathway for metastatic activity have been identified and functionally validated.

TRIM21 is a novel regulator of Par-4 in colon and pancreatic cancer cells.

The prostate apoptosis response protein 4 (Par-4) is a tumor-suppressor that has been shown to induce cancer-cell selective apoptosis in a variety of cancers. The regulation of Par-4 expression and activity is a relatively understudied area, and identifying novel regulators of Par-4 may serve as novel therapeutic targets. To identify novel regulators of Par-4, a co-immunoprecipitation was performed in colon cancer cells, and co-precipitated proteins were identified by mass-spectometry. TRIM21 was identified as a novel interacting partner of Par-4, and further shown to interact with Par-4 endogenously and through its PRY-SPRY domain. Additional studies show that TRIM21 downregulates Par-4 levels in response to cisplatin, and that TRIM21 can increase the resistance of colon cancer cells to cisplatin. Furthermore, forced Par-4 expression can sensitize pancreatic cancer cells to cisplatin. Finally, we demonstrate that TRIM21 expression predicts survival in pancreatic cancer patients. Our work highlights a novel mechanism of Par-4 regulation, and identifies a novel prognostic marker and potential therapeutic target for pancreatic cancer.

Osteopontin identified as lead marker of colon cancer progression, using pooled sample expression profiling.

BACKGROUND: New tumor markers and markers of tumor progression are needed for improved staging and for better assessment of treatment of many cancers. Gene expression profiling techniques offer the opportunity to discover such markers. We investigated the feasibility of sample pooling strategy in combination with a novel analysis algorithm to identify markers. METHODS: Total RNA from human colon tumors (n = 60) of multiple stages (adenomas; cancers with modified Astler Collier stages B, C, and D; and liver metastases) were pooled within stages and compared with pooled normal mucosal specimens (n = 10) by using oligonucleotide expression arrays. Genes that showed consistent increases or decreases in their expression through tumor progression were identified. Northern blot analysis was used to validate the findings. All statistical tests were two-sided. RESULTS: More than 300 candidate tumor markers and more than 100 markers of tumor progression were identified. Northern analysis of 11 candidate tumor markers confirmed the gene expression changes. The gene for the secreted integrin-binding protein osteopontin was most consistently differentially expressed in conjunction with tumor progression. Its potential as a progression marker was validated (Spearman's rho = 0.903; P<.001) with northern blot analysis using RNA from an independent set of 10 normal and 43 tumor samples representing all stages. Moreover, a statistically significant correlation between osteopontin protein expression and advancing tumor stage was identified with the use of 303 additional specimens (human cancer = 185, adenomas = 67, and normal mucosal specimens = 51) (Spearman's rho = 0.667; P<.001). CONCLUSIONS: Sample pooling can be a powerful, cost-effective, and rapid means of identifying the most common changes in a gene expression profile. We identified osteopontin as a clinically useful marker of tumor progression by use of gene expression profiling on pooled samples.

Increased Src activity disrupts cadherin/catenin-mediated homotypic adhesion in human colon cancer and transformed rodent cells.

Src has been implicated in the development and progression of human colon cancer. Because the capacity for tumor cells to dissociate from the primary tumor is a critical step in the development of metastases, the effect of a naturally occurring, activated Src-531 on intercellular adhesion was examined. Homotypic adhesion was assessed using dissociation assays on Src-transformed rat fibroblasts and human colon cancer cell lines. The data indicate that both rodent and human cells expressing the mutant Src protein display up to 7-fold less homotypic adhesion than do wild-type cells (P < 0.01). Experiments demonstrated that cadherin was phosphorylated in cells transfected with activated Src and that cadherin/catenin complexes were disrupted as a result. Experiments using dominant negative (DN) Src or an Src-specific inhibitor (PD 180970), demonstrated that adhesion was restored when Src activity was inhibited in Src-531 transfectants, confirming that Src is a causal factor in the decreased homotypic adhesion observed. In addition, DN Ras, DN focal adhesion kinase (FAK), but not Stat3beta, restored intercellular adhesion, which suggested that Ras and FAK may be downstream effectors of Src-mediated homotypic adhesion. Collectively, these data support a role for Src, Ras, and FAK in the regulation of intercellular adhesion, which may in turn regulate metastatic potential of human colon cancer cells.

Regulation of caspase expression and apoptosis by adenomatous polyposis coli.

The adenomatous polyposis coli (APC) gene, a member of the WNT pathway, has been shown to assign intestinal epithelial cells to a program of proliferation or differentiation through regulation of the beta-catenin/TCF-4 complex. Wild-type APC, in certain cellular contexts, appears to induce differentiation and apoptosis, although mutant forms of APC, known to produce polyps and ultimately cancers, may suppress these events. Here, we show that mutant forms of APC can induce repression of select terminal caspases as a potential means of attenuating responses to apoptotic stimuli. Using gene expression profiling to interrogate the intact intestines of Apc(+/min) mice harboring numerous polyps, we identified a reduction in the mRNA expression of both caspases 3 and 7. We additionally identified a reduction in protein levels of caspase-3, caspase-7, and caspase-9 in human colon cancer specimens known to harbor APC mutations. A reduction in caspase protein levels resulted in resistance to apoptotic-inducing agents and restoration of caspase levels reinstated apoptotic capacities. Consistent with Wnt pathway involvement, dominant negative TCF/LEF induced caspase protein expression. These data provide support for the hypothesis that one of the functions of APC is the regulation of caspase activity and other apoptotic proteins by controlling their expression levels in the cell.

Identification of Src transformation fingerprint in human colon cancer.

We used a classical rodent model of transformation to understand the transcriptional processes, and hence the molecular and cellular events a given cell undergoes when progressing from a normal to a transformed phenotype. Src activation is evident in 80% of human colon cancer, yet the myriad of cellular processes effected at the level of gene expression has yet to be fully documented. We identified a Src 'transformation fingerprint' within the gene expression profiles of Src-transformed rat 3Y1 fibroblasts demonstrating a progression in transformation characteristics. To evaluate the role of this gene set in human cancer development and progression, we extracted the orthologous genes present on the Affymetrix Hu95A GeneChip (12k named genes) and compared expression profiles between the Src-induced rodent cell line model of transformation and staged colon tumors where Src is known to be activated. A similar gene expression pattern between the cell line model and staged colon tumors for components of the cell cycle, cytoskeletal associated proteins, transcription factors and lysosomal proteins suggests the need for co-regulation of several cellular processes in the progression of cancer. Genes not previously implicated in tumorigenesis were detected, as well as a set of 14 novel, highly conserved genes with here-to-fore unknown function. These studies define a set of transformation associated genes whose up-regulation has implications for understanding Src mediated transformation and strengthens the role of Src in the development and progression of human colon cancer. Supportive Supplemental Data can be viewed at http://pga.tigr.org/PGApubs.shtml.

Peroxisome proliferator-activated receptor alpha induces rat sterol carrier protein x promoter activity through two peroxisome proliferator-response elements.

Sterol carrier protein x (SCPx) plays a critical role in the peroxisomal oxidation of fatty acids. It has been previously demonstrated in streptozotocin-induced diabetic rats that SCPx expression is induced in association with an elevation in serum fatty acid and triglyceride levels. To elucidate the mechanisms underlying the expression of this gene during diabetes, the rat SCPx promoter was cloned and analyzed for regulatory motifs. Sequence analysis of this TATA-less promoter revealed two putative peroxisomal-proliferator-response element (PPRE) binding motifs at positions -134 and -869 relative to the translation start site. To examine peroxisomal-proliferator-activated receptor alpha (PPARalpha) effects on this gene, 935 bp of the SCPx promoter containing both PPRE motifs was cloned in front of the chloramphenicol acetyl-transferase gene or the luciferase gene and co-transfected into HTB-9 cells with vectors that encoded for PPARalpha and retinoid X receptor (RXR). The results indicate that PPARalpha was able to induce SCPx promoter activity in both cases, an effect that was enhanced by RXR and clofibrate. In addition, mutational analysis studies demonstrated that both PPREs contributed to the PPARalpha/RXRalpha-dependent activation of the SCPx promoter. Mobility shift assays and supershift analysis showed that nuclear extracts containing PPARalpha bound to the two PPRE motifs. This investigation indicates that similar to other genes involved in beta-oxidation, SCPx transcription may be controlled by fatty acid levels via PPARalpha.

Osteopontin identified as colon cancer tumor progression marker.

Identifying molecular markers for colon cancer is a top priority. Using a pooled sample approach with Affymetrix GeneChip technology, we assayed colon cancers derived from a series of clinical stages to identify molecular markers of potential prognostic value. Of 12000 genes assessed, osteopontin emerged as the leading candidate tumor progression marker. Osteopontin is a secreted glycoprotein known to bind integrins and CD44. Its actual molecular function remains elusive but its increased expression correlates strongly with tumor progression.

Par-4 as a potential target for cancer therapy.

INTRODUCTION: Despite extensive research, cancer continues to be a leading cause of death worldwide and is expected to continue to rise as a result of an aging population. Therefore, new therapies are constantly being developed. Par-4 is a naturally occurring tumor suppressor protein that is capable of inducing apoptosis in cancer, but not normal cells. For this reason, Par-4 offers an attractive target for development of cancer therapy, particularly of difficult to treat cancers. AREAS COVERED: The mechanisms by which Par-4 induces cell death are summarized. The ways that Par-4 is controlled in cancer cells are discussed. We discuss how different research groups have developed ways to overexpress and/or activate Par-4 in vitro and in vivo. The studies described demonstrate that when Par-4 levels and/or activity are increased, susceptibility to apoptosis is enhanced and tumor growth is inhibited. EXPERT OPINION: Par-4 is a promising therapeutic protein that can be overexpressed and/or activated to induce apoptosis in a cancer-selective manner. This cancer selectivity is important given that the side-effects of chemotherapeutics can be as debilitating as cancer itself. However, there are key issues that need to be addressed to optimize the effects of Par-4 in patients.

The Akt inhibitor ISC-4 synergizes with cetuximab in 5-FU-resistant colon cancer.

Phenylbutyl isoselenocyanate (ISC-4) is an Akt inhibitor with demonstrated preclinical efficacy against melanoma and colon cancer. In this study, we sought to improve the clinical utility of ISC-4 by identifying a synergistic combination with FDA-approved anti-cancer therapies, a relevant and appropriate disease setting for testing, and biomarkers of response. We tested the activity of ISC-4 and 19 FDA-approved anticancer agents, alone or in combination, against the SW480 and RKO human colon cancer cell lines. A synergistic interaction with cetuximab was identified and validated in a panel of additional colon cancer cell lines, as well as the kinetics of synergy. ISC-4 in combination with cetuximab synergistically reduced the viability of human colon cancer cells with wild-type but not mutant KRAS genes. Further analysis revealed that the combination therapy cooperatively decreased cell cycle progression, increased caspase-dependent apoptosis, and decreased phospho-Akt in responsive tumor cells. The synergism between ISC-4 and cetuximab was retained independently of acquired resistance to 5-FU in human colon cancer cells. The combination demonstrated synergistic anti-tumor effects in vivo without toxicity and in the face of resistance to 5-FU. These results suggest that combining ISC-4 and cetuximab should be explored in patients with 5-FU-resistant colon cancer harboring wild-type KRAS.

Seroreactivity to LGL leukemia-specific epitopes in aplastic anemia, myelodysplastic syndrome and paroxysmal nocturnal hemoglobinuria: results of a bone marrow failure consortium study.

Large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of antigen-activated cytotoxic T cells (CTL). Patients frequently exhibit seroreactivity against a human T-cell leukemia virus (HTLV) epitope, BA21. Aplastic anemia, paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome are bone marrow failure diseases that can also be associated with similar aberrant CTL activation (LGL-BMF). We identified a BA21 peptide that was specifically reactive with LGL leukemia sera and found significantly elevated antibody reactivity against the same peptide in LGL-BMF sera. This finding of shared seroreactivity in LGL-BMF conditions and LGL leukemia suggests that these diseases might share a common pathogenesis.

The Akt inhibitor ISC-4 activates prostate apoptosis response protein-4 and reduces colon tumor growth in a nude mouse model.

PURPOSE: Prostate apoptosis response protein-4 (Par-4) sensitizes cells to chemotherapy; however, Akt1 inactivates Par-4. Previously we showed that Par-4-overexpressing colon cancer cells responded more readily to 5-fluorouracil (5-FU) than their wild-type counterparts. In this study we investigated (i) the effects of the Akt inhibitor, phenylbutyl isoselenocyanate (ISC-4), on tumor growth in nude mice and (ii) bystander effect of Par-4-overexpressing cells on wild-type tumor growth. EXPERIMENTAL DESIGN: Mice (n = 80) were injected with wild-type HT29 human colon cancer cells in the right flank. Forty of the mice were also injected in the left flank with HT29 cells engineered to overexpress Par-4. The mice were treated with 5-FU, ISC-4, a combination, or vehicle. RESULTS: ISC-4 reduced tumor growth, with or without 5-FU. When Par-4-overexpressing tumors were present, wild-type tumors grew more slowly compared to when no Par-4-overexpressing tumors were present. The level of Par-4 protein as well as the Par-4 binding protein, GRP78, was increased in wild-type cells growing in the same mouse as Par-4-overexpressing tumors compared with wild-type tumors growing without Par-4-overexpressing tumors. CONCLUSIONS: Par-4-overexpressing tumors exhibited a bystander effect on wild-type tumors growing distally in the same mouse. This suggests that gene therapy need not achieve total penetration to have a positive effect on tumor treatment. Inhibition of Akt with ISC-4 inhibited tumor growth and had a greater effect on cells overexpressing Par-4. The data indicate ISC-4 alone or in combination with Par-4 can greatly reduce tumor growth.