Based on these observations we surmise that similar HDACI induced gene research use only networks were uncovered by IPA and KEGG analyses. A putative involvement of MAPK pathways in the action of pan HDAC inhibitors The network analyses of genes that were differentially regulated by CBHA and TSA, regardless of whether it was done by IPA or KEGG programs, strongly predicted a role of PTEN PI3K AKT PKB and MAPK signaling pathways in the actions of HDACIs. We reported earlier that both CBHA and TSA potently induced the expres sion of PTEN and concomitant reduction in PI3K and AKT phosphorylation in H9c2 cells as well as in the in tact heart. To test a potential role of MAP kinases, we extracted proteins from H9c2 cells incubated with CBHA or TSA for various time intervals and assessed the steady state levels of total and phosphorylated ERK, JNK and p38 MAPK.

As shown in Figure 11, an expos ure to TSA for 4h led to a reduced phosphorylation of ERK and its phosphorylation remained inhibited until 24h. TSA treatment also significantly suppressed phosphorylation of p38 as early as 2h. Finally, an exposure of H9c2 cells to CBHA resulted in a reduction of pERK at 4h, while the levels of p p38 kinase were not significantly affected by CBHA. The temporal changes in the regulation of JNK in response to CBHA or TSA were inconclusive. Finally, it should be noted that neither TSA nor CBHA altered the steady state levels of total ERK or p38 kinases.

Frequency of putative transcription factor binding sites in differentially expressed genes in response to CBHA and TSA With an aim to elucidate potentially common pathways involved in the induction of genes by CBHA and TSA, we extended gene network analyses by an in silico exam ination of transcription factor binding sites in the promoters of DEGs. We explored 1 kb of DNA upstream of transcription start site of all differentially expressed genes by CORE TF, a web based program that identifies dominant TFBS. As shown in Table 5, in DEGs induced by CBHA at 6 and 24h, the topmost transcrip tional factor motifs were those of AP2, CHCH, E2F1, EGR2 and ETF. An over representation of AP2, CHCH, E2F1, EGR2 and ETF was also seen in TSA treated cells, additionally, the promoters of the TSA induced DEGs expressed zinc finger containing transcription factors. Finally, NF Y specific motifs were overrepresented in DEGs induced by TSA at 24h.

The preponderance of E2F1, EGR2, Sp1 and KROX tran scription factor binding sites in the DEGs induced by ei ther pan HDAC inhibitor was consistent with an ability of these transcription factors to regulate genes involved in cell proliferation and apoptosis. The members of the E2F family, that bind to RB1, also Dacomitinib play a key role in regulating G to S transition, similarly, NF Y has a fun damental role in the expression of genes that regulate G2 M phase of the cell cycle.

Statistical analysis of miRNA array data There were 664 miRNAs profiled for each of the 40 sam ples, Ct values were obtained with the automatic baseline and manual Ct set to 0. 1 threshold. Some miRNAs were only minimally expressed, http://www.selleckchem.com/products/ganetespib-sta-9090.html and were excluded from further analyses, specifically we excluded those for which 20% or more of the samples had a missing Ct or Ct 35. Lowess smoothing was used to normalize measures across individuals. Missing values were imputed using a K Nearest Neighbour approach as described by Tusher et al. Any particularly extreme values for each miRNA were shrunk in towards the center of the distribution so as to lessen their influence. For each comparison of two groups, two sample t tests were used to assess nominal significance.

The Westfall Young min P approach using 1000 permutations of group labels was used to obtain p values adjusted for multiple testing. Empirical q values were also estimated using the permuted data. Heat Maps and Box plots based on the miRNA array data Normalized Ct values were adjusted by subtracting the Ct value from an arbitrary constant value of 40 so that a higher adjusted Ct value would correspond to a higher miRNA expression. The table of adjusted Ct values for the 20 significantly dysregulated miRNAs between PGRN and PGRN FTLD TDP patients was loaded in Cluster 3. 0. A heat map showing the miRNA expression profiles for all the samples was gen erated after median centering the adjusted Ct values for each miRNA. The normalized and adjusted Ct values were summarized across groups with boxplots.

Validation of miRNA candidates in frontal cortex and cerebellum The top 20 miRNA candidates identified in the miRNA array experiment were selected for validation by qRT PCR in the same set of 8 PGRN and 32 PGRN FTLD TDP patient samples. In brief, 50 uls of reverse transcrip tion primers for the 20 miRNAs plus RNU48 as an endogenous control were divided into 3 primer pools, lyophilized, and subse quently resuspended in water for each pool resulting in 5�� multiplex RT primer pool. Total RNA was reverse transcribed in a 20 ul reaction volume using the miRNA Reverse Transcription Kit and 1 ul of cDNA was used in the Taqman miRNA assays. Where duplicate Ct values differed by more than 2, the more extreme one relative to the distribution of Ct values across all samples was deleted, otherwise the mean of the duplicates was used as the final Ct for a tran script.

Delta Cts were calculated by subtracting the Ct of the endogenous control RNU48. Minus delta Cts were used as the final Dacomitinib values for analysis and assumed to represent the log base 2 of scaled expression levels. Two sample t tests and corresponding 95% confidence inter vals were used to compare groups, and the differ ences between means and CIs were exponentiated to provide fold change estimates under the assumption of perfect probe efficiency.

Silencing p21 prevents breast tumor local invasion in vivo and cancer cell migration and invasion in vitro To investigate the contribution Nutlin-3a buy of p21 to tumor formation and progression in breast cancer, we used a bone meta static cell line SCP2, a sub progeny of the human triple negative breast cancer MDA MB231 cells. We first assessed the effect of suppres sing p21 on tumor growth using a mammary fat pad xeno graft mouse model. A specific p21 shRNA was stably transfected to generate a pool of p21 deficient SCP2 cells. Knockdown of p21 using shRNA efficiently reduced p21 protein expression, as compared to parental SCP2 cells. Parental and shRNA p21 SCP2 cells were orthotopically injected into the mammary fat pad of female Balb/c nude mice. Tumor growth was monitored weekly.

There was no difference in the rate of primary tumor formation or tumor size between animals injected with parental or p21 deficient cells, suggesting p21 is not likely involved in tumor formation. Next, we evaluated the effect of p21 depletion on tumor invasiveness, a critical step for early tumor progression. Intact tumors were taken with the overlaying skin and surrounding deep tissues and analyzed by a pathologist. Tumor invasiveness was assessed by determining the extent of infiltration of cancer cells to the surrounding tissue, as previously described. As shown in Figure 2C, tumors from the parental SCP2 group dis played no clear margin with the surrounding tissues and were deeply invading into nearby structures.

In contrast, tumors derived from animals transplanted with p21 depleted SCP2 cells formed a well encapsulated tumor mass that did not invade the surrounding tissues, strongly suggesting that p21 plays an important role in tumor invasion. This was confirmed in vitro, as p21 gene silencing in SCP2 cells inhibited both cell migration and invasion. As shown in Figure S2A, none of the animals in which parental or p21 depleted SCP2 cells were injected into the mammary fat pad developed any bone lesions after two months, the date at which mice had to be sacrificed due to the tumor size. This timing may have been insufficient for tumor cells to grow into visible distant lesions in the mouse. Thus, to investigate whether p21 is involved in the later stage of breast cancer progression, we examined its involvement in the development of bone osteolytic lesions using an intratibia injection model of parental and p21 deficient SCP2 cells in female Balb/c nude mice. By by passing the early steps of metastasis, this experi mental model allows for the assessment of tumor cell metastasis and survival in the bone Anacetrapib marrow.

7 cells plated in 6 cm dishes were pre incubated in serum free DMEM for 2 h before stimulated with PS F2. At various time after stimulation, whole cell lysates were prepared by treating cells with 200 ul of SDS PAGE sample buffer. To prepare cytoplasmic and nuclear extracts, cells were STI 571 harvested and resuspended in 150 ul of hypotonic buffer and incubated on ice for 15 min. The samples were then mixed with 10 ul of 10% NP 40 and centrifuged at 16,000 g for 30 sec. The supernatant representing the cytosolic fraction was collected, and the pellet containing the nuclei was resuspended in 50 ul of nuclear extract buffer and incubated at 4 C for 15 min with vigorous shaking. After centrifugation at 16,000 g for 5 min, the supernatant representing the nuclear fraction was collected and stored at ?20 C.

Western blot analysis Cell lysates in SDS PAGE sample buffer were heated at 95 C for 5 min, separated by 12. 5% SDS PAGE, and transferred to a nitrocellulose membrane. The mem brane was blocked with 5% bovine serum albumin in Tris buffered saline containing 0. 05% Tween 20 and incubated with primary antibodies specific for JNK, p38, ERK, phospho JNK, phospho p38, phospho ERK, NF ��B, I ��B, B actin, or histone deacetylase 1 at 4 C for overnight. The mem brane was then incubated with horseradish peroxidase conjugated secondary antibodies and visualized with an enhanced chemiluminescence kit and a chemiluminescence imaging system. Densitometric analysis of band in tensities was performed using the ImageJ software. Statistical analysis Statistical analysis was performed using an unpaired, two tailed Students t test and a P 0.

05 was considered significant. Data are reported as mean and SEM. Background Oral cancers are malignancies arising from either tongue, lip, gingivae, palate, salivary glands, buccal mu cosa or floor of the mouth, and accounts for an esti mated 2. 08% of total cancer cases worldwide in 2011. About 90% of oral cancers are squamous carcinomas, with main treatment options that in clude surgery followed by radiotherapy and adjuvant chemotherapy. Even though oral cancers are rela tively preventable, diagnosed patients often face a low five year survival rate of 58%, which has remained un changed over the past three decades despite recent treat ment advances.

Presently, platinum based drugs such as cisplatin, remains one of the most commonly used chemotherapeutic agents available for the treat ment of advanced oral cancers. While CDDP treat ment often results in initial responses and disease stabilization, its long term success is hindered by the de velopment of drug Dacomitinib resistance and dose limiting toxicities through the occurrence of DNA cross linking in sur rounding non cancerous cells. Thus, there is an on going need for modified CDDP combination regimes that can ideally reduce overall dose toxicity through chemo sensitization of oral cancer cells.

showed that valproic acid potentiated the sensitivity of prostate cancer selleck inhibitor cells to cisplatin through down regulation of HR repair and DNA damage response genes such as BRCA1. The decrease in BRCA1 gene transcription was due to a reduction in binding of the activating protein, E2F1, to the BRCA1 promoter. In the same prostate cancer cell line model, a new HDAC inhibitor, H6CAHA, sup pressed the expression of BRCA1 mRNA, and when used in combination with g radiation, prevented the growth of tumor xenografts. The sensitizing properties of HDAC inhibitors to DNA damaging agents has been linked to aberrant dou ble strand break repair and cellular stress signaling. The present study confirms reports that HDAC inhibi tion, in combination with DNA damaging agents, increases the phosphorylation of H2A.

X, a known mar ker of DNA double strand breaks. A study con ducted in a metastatic breast cancer cell line provides evidence of increased phosphorylation of H2A. X and enhanced sensitivity to vorinostat in combination with radiation. In both human glioma and prostate can cer cells, vorinostat reduced DNA dependent protein kinase and Rad 51, two critical components of DNA double strand break repair machinery. In the human melanoma cell line, A375, vorinostat sensi tized cells to radiation induced apoptosis by inhibiting key DNA repair genes, Ku70, Ku80 and Rad 50. Using cDNA expression arrays, phenylbutyrate attenu ated the expression of DNA PK and worked synergisti cally with ionizing radiation to induce apoptosis in prostate cancer cell lines.

BRCA1 has many diverse functions in the cell includ ing transcriptional control through modulation of chro matin structure as BRCA1 is known to interact with the SWI/SNF chromatin remodeling complex. The BRCA1 SWI/SNF complex is believed to be essential for the activation of genes involved in the DNA damage response and this complex has a direct role in HR by enabling access to sites of DNA damage. The BRCA1 C terminal domain of the BRCA1 protein associ ates with both HDAC1 and HDAC2, and prior studies suggest that this association directly represses transcrip tion. In this study, the ChIP assay demonstrated that the amount of BRCA1 promoter DNA containing acetylated histones was decreased following M344 and cisplatin combination treatment relative to controls.

This result suggests that BRCA1 is not a direct target of M344 activity, but that M344 may enhance the expres sion or activity of a transcriptional repressor of BRCA1. As an example, the Inhibitor of DNA binding 4 is a dominant negative transcriptional regulator, which has been shown to repress the BRCA1 promoter. Studies have identified an inverse correlation between ID4 and BRCA1 mRNA and protein AV-951 expression levels in breast and ovarian tumour tissue. Further studies are needed to evaluate ID4s role in BRCA1 transcrip tional activity and as a potential marker of BRCA1 expression.

This compound caused inhibition of HDAC 8 activity with no effect on the activity of HDAC 1/2. The protein levels of HDAC 2, 3 and 8 were found to be drastically reduced with no change in HDAC 4 6 upon chrysin treat ment. Chrysin caused histone modifications such selleck chem as acetylation and methylation at p21 promoter particu larly at STAT binding site and resulted in increased p21 promoter activity. More over chrysin as a HDAC inhibitor cause apoptosis by decreasing the levels of NF kB targeted and HDACi related genes such as Bcl xL, survivin and increased the level of caspase 3 proteins. Methods Chemical structure and extraction of natural compounds The dried stem bark of dundilum tree, Oroxylum indicum was grinded and extracted consecutively with hexane in a soxhlet apparatus.

Solid residue in the hexane extract was filtered and subjected to silica gel column chromatography to isolate two major frac tions. Fraction F1 was purified on silica gel column chromatography eluted with 0. 5 % MeoH in Chloroform to isolate methoxy chrysin. Simi larly, Fraction F2 was subjected to repeated column chro matography with the elution of 2 % MeoH in Chloroform to isolate oroxylin A and chrysin. The purifi cation, chemical structure and characterization of all three compounds were determined via extensive spectroscopic NMR, ESI MS, and HPLC methods. The conserved methyl oxide and hydroxyl group are shown in the chemical struc ture of small flavonoid compounds. Cell culture A375, U3A cell lines were maintained in DMEM. Whereas K562 cell line was maintained in RPMI media.

All three cell lines were supplemented with 10 % FCS, 1 % pencillin/ streptomycin 5 % glutamine. These cell lines were grown at 370 C in a humidified chamber containing 5 % CO2. MTT assay Cell viability was assessed by the MTT assay, a mitochon drial function assay. It is based on the ability of viable cells to reduce the MTT to insoluble formazan crystals by mitochondrial dehydrogenase. A375 cells were seeded in a 96 well plate at a density 10,000 cells/well. After overnight incubation, cells were treated with compounds chrysin, methoxy chrysin, oroxylin A at a final concentration of 40 uM and Trichostatin A at a final concentration of 4 uM and incubated for 24 h. Medium was then discarded and replaced with 10 uL MTT dye. Plates were incubated at 37 C for 2 h.

The resulting formazan crystals were solu bilized in 100 uL extraction buffer. The optical density was read at 570 using micro plate reader. Cell Cycle Analysis 5 X 105 A375 cells were seeded in 60 mm dish and were allowed to grow for 24 h. Compounds chrysin, oroxylin A, methoxy chrysin at 40 uM final concentration as well as TSA at 4 uM final concentration were added Anacetrapib to the culture media, and the cells were incubated for an additional 24, 48 and 72 h.

For 41 out of the 45 interactors tested specific fluorescence was observed upon addition of the VC155 Hoxa1 fusion protein. Distinct patterns of intracellular interactions were observed. For 31 proteins, interactions took place in the nucleus. Of these, 16 proteins appeared to contact Hoxa1 exclusively in the nucleus, while 15 also displayed other patterns of subcellular fluorescence complementation. selleck chemicals llc Among the proteins found to bind Hoxa1 in the nucleus, some were known transcription factors or were known to have nuclear functions, but other were not. A set of proteins shared a similar interaction pattern characterized by a diffuse, finely punctuated cytoplasmic signal without nuclear staining. This subcellular localization pattern was observed for different proteins reported to participate in a common signaling pathway.

Examples are TRAF, TRIP or PDCD6IP which are found asso ciated with the TNFR family of receptors, SPRY1 and PDCD6IP modulating RTK downstream signaling, PDLIM7 and RBPMS which are involved in the BMP TGFB sig naling regulation and LPXN, PDCD6IP and TRIP6 known to associate with focal adhesion sites and related signal transduction. As a control, in cells co expressing GST TRAF1 fusion and wildtype Hoxa1, proteins displayed an overlapping intracellular distribution consistent with the BiFC signal observed with VN173 TRAF1 VC155 Hoxa1. Fourteen interactors tested displayed variable interaction patterns, showing mostly nuclear to nuclear and cytoplasmic or nuclear and vesicular BiFC signal. This heteroge neous distribution suggests a coordinated shuttling be tween cell compartments for Hoxa1 and some partners.

The specific associations between Hoxa1 and 41 interactors detected by BiFC shows that Hoxa1 can associate dynamically with distinct categories of proteins in distinct intracellular domains. Discussion By a high throughput Y2H screen we identified 59 Hoxa1 interacting proteins among which 45 were con firmed by co precipitation from animal cells. The intra cellular localization of 41 interactions was further detected by a BiFC approach. This is the first exhaustive screen and analysis for interactors of a Hox protein. Our data support the conclusion that Hox proteins, and Hoxa1 in particular, known as crucial transcription factors controlling developmental processes can fulfill unexplored roles in cell signaling, cell adhesion, or ves icular trafficking.

Hoxa1 appears to interact with several proteins found to be part of molecular platforms associated with a few signaling pathways, membrane dynamics and ves icular trafficking. These platforms contact activated receptors at the plasma membrane and can positively or negatively modulate the downstream signal ing or subsequent internalization in the endosomal Cilengitide com partment.

Consequently, the delayed administration of MK 8776 provides greater tumor growth delay in xeno graft models. These results have important implications for the design of clinical trials of this drug combination. Background Pancreatic cancer is one of the such information most aggressive human malignancies, with less than 5% of patients still alive five years after diagnosis. In 2012, it is estimated that a total of 43,920 patients will be diagnosed with pancreatic cancer in the United States, and 37,390 will die of this disease. Pancreatic cancer is characterized by a rapid disease progression and highly invasive phenotype. Most patients are with unresectable tumor at the time of diag nosis, leaving chemotherapy and radiation as the only available treatment options.

For the past decades, gemcitabine has been the standard treatment for advanced pancreatic cancers, prolonging survival by 5 6 months. However, a large percentage of pancreatic cancers do not respond to gemcitabine, probably due to the high level of intrinsic and acquired chemo resistances. Angiogenesis is essential for tumor growth and metas tasis. Tumor associated angiogenesis is critical for pan creatic cancer progression. Several modes of vessel formation have been proposed so far vasculogenesis, angiogenesis, intussusceptions, vascular cooption and vas culogenic mimicry. VM is the process where fluid conducting channels were formed by the highly inva sive and genetically dysregulated tumor cells. Tumors with high VM abilities are often highly aggressive and associated with poor prognosis.

VM has been observed in a variety of aggressive tumors including carcinomas, breast cancers, liver cancers, ovarian can cers, prostate cancers, sarcomas, gliomas and melano mas. Pancreatic cancer represents one of the most vascularized and angiogenic solid tumors. In the current study, we found that many human pancre atic cancer cells could also form tube like structure in vitro. In the current study, we aimed to seek novel and more efficient treatment strategies by targeting angiogenic mim icry in pancreatic cancer cells. Suberoylanilide hydroxamic acid belongs to the histone deacetylases inhibitors, which represent a new class of anti cancer therapeutics. Studies have confirmed its high effi ciency in inhibiting angiogenesis in pre clinical animal models and early phase clinical trials.

SAHA in hibits the in vitro and in vivo growth of transformed hu man cancer cells, including prostate, bladder and ovarian tumor cells. SAHA has been tested in phase I and phase II clinical trials for the treatment of various malig nancies, and has demonstrated significant anti cancer effi ciency at well tolerated doses. Meanwhile, Carfilzomib studies have shown that SAHA exhibits profound inhibitory effects against human pancreatic cancer cells.

Analysis of intracellular signaling The interference selleckbio of RAD001, AEE788 or VPA with intracellular signaling was investigated. VPA diminished EGFr, pERK and phosphorylated p70S6k in all cell lines. Analysis of pAkt revealed conflicting results, since this protein was dis tinctly reduced in DU 145, strongly enhanced in LNCaP, whereas a protein double band appeared in PC 3 cells. Both, pEGFr and pERK were down regulated in all tumor cells following AEE788 exposure, but pp70S6k expression was similar between treated and untreated cells. The latter was also true with respect to pAkt. RAD001 reduced pEGFr in PC 3 and LNCaP and pERK in PC 3 and DU 145 cells. RAD001 also down regulated pp70s6 k in all explored cell lines. Triple drug treatment provided combinatorial benefit with respect to EGFr, pEGFr, pERK and pp70S6k loss.

Furthermore, the amount of pAkt proteins was greatly but not in PC 3 cells. Cyclin E was elevated by VPA but reduced by AEE788. RAD001 profoundly altered cyclin B in DU 145 but not in PC 3 and LNCaP cells. Several investigators have recently demonstrated that a tumor cells response to a particular drug depends on receptor and protein configuration, which is characteristic in the different PC cell lines. It has been shown that the PC phenotype determines its sensitivity towards treatment with a tyrosine kinase inhibitor, mTOR or HDAC inhibitor. The variable response of the cell lines to a single drug treatment is not foreseeable, due to the PCs heteroge neous nature, resulting in different malignant matura tion pathways and protein profiling.

Analysis of mTOR in PC patients revealed distinct heterogeneity in the study cohort. The same was true with respect to EGFr and VEGF expression, and to the HDAC level. Given the molecular specificity of each targeted compound, it is unrealistic to expect similar biochemical reactions in every PC cell line. The data presented here demonstrate that the triple drug combination circum vents this problem by exerting anti cancer properties in different tumor cell types according to the particular molecular profile. From a clinical viewpoint, simulta neous use of a set of drugs with complementary phar macological characteristics may enhance the total percentage of responders, as well as the elimination rate of tumor clones in each individual patient.

The VPA RAD001 AEE788 drug combination diminished cdk1, cdk2, cdk4 and cyclin B in PC 3, DU 145 and LNCaP elevated in PC 3 and LNCaP cells, exceeding Cilengitide the pAkt levels evoked by single drug use. pEGFr down regulation induced by single drug treatment in PC 3 and LNCaP cells was reverted by the triple drug application. Discussion The combined inhibition of EGFr VEGFr and mTOR related pathways, coupled with HDAC deactivation, pro foundly blocked PC growth and adhesion. The blocking effect was similar in all employed cancer cell lines and more extensive, compared to the single drug regimen.

Since adequate expression of costimulatory adhesion molecules is essential for proper T cell responses, be it cell survival, activation or proliferation, we decided to analyze the expression levels of various cytokine receptors, cell adhesion molecules, and cell surface receptors in a cell population treated with fairly TSA. Splenocytes from C57BL 6 mice were stimulated with sol uble anti CD3 antibody or IL 2 and exposed to 100 nM TSA. Samples were taken after 4 and 20 hours, and expression of cell surface markers examined by flow cytometry. CD4 T cells stimulated with IL 2 and exposed to TSA for 4 hours, exhibited lower levels of expression of CD11c, CD69, CD28, CD40, and CD40L, and higher levels of expression of IL2R with all others remaining constant.

After 20 hours of exposure IL2R, ICAM 1, CD8a, CD11c, CD45RB, CD69, CD28, CD40L, CD80, CD86 and CD95 were downregulated to various extents, but none of the examined molecules was upregulated. In non CD4 cells stimulated with IL 2 and exposed to TSA for 4 hours, the pattern of effects was very similar. Thus, expression of CD11c, CD69, CD28, CD40, and CD40L was downregu lated, whereas expression of IL2R was increased. After 20 hours of exposure, IL2R, IL2R, LFA 1, ICAM 1, CD8a, CD11b, CD11c, CD45RB, CD69, CD28, CD40, CD40L, CD48, CD86, and CD95 were downregulated to various extents, but of the examined molecules only CD80 was upregulated. Stimulation of PBMCs with anti CD3 led to profound alterations in the profile of TSA mediated cell surface molecule expression.

CD4 T cells exposed to TSA for 4 hours, exhibited higher levels of expression of CD11c and ICAM 1 with all others remaining constant. After 20 hours of exposure, IL2R, IL2R, LFA 1, CD45RB, CD28, CD40, CD40L, and CD48 were down regulated, whereas ICAM 1 and CD69 were upregulated. In non CD4 cells treated with TSA for 4 hours levels of expression of CD11c were increased. After 20 hours of exposure, IL2R, IL2R, LFA 1, ICAM 1, CD8a, CD11c, CD28, CD40L, and CD95 were downregulated, and CD11b, CD40, and CD86 were upregulated. These results demonstrate that HDAC inhibitors may modulate T cell function in part through changes in the expression of cell surface proteins. The differences in expression of cell surface molecules, such as CD95 Fas, in PBMCs in the two cell subpopula tions we examined suggested to us that the various cell types might be affected to different extents by TSA.

To clarify this point, we cultured PBMCs in the presence of either soluble anti CD3 antibody or IL 2 and treated with TSA or DMSO for 20 hours. Subsequently, cells were analyzed by flow cytometry to determine cell viabil ity and the ratio of CD4 T cells versus non CD4 cells. Plotting of the percentage of total viable cells in the pop ulation shows that TSA induces apoptosis in PBMCs Brefeldin_A to a similar extent in anti CD3 or IL 2 stimulated cells.