Prevalence of the BCR/ABL fusion gene and T cell stimulation capacity of dendritic cells in chronic myelogenous leukemia.

Dendritic cell (DC) vaccines are promising for immunotherapy, and their production using CD34+ hematopoietic stem cells (HPSCs) from patients with chronic myelogenous leukemia (CML) and healthy donors is well established. However, the generation of CD1a+CD14- DCs and their functional properties in patients with CML remain elusive. Here, we aimed to study the biology of DCs generated from CD34-/low HPSCs and evaluate the status of their BCR/ABL translocation, ability to stimulate T cells, and capacity of endocytosis compared to DCs derived from CD34+ HPSCs from both patients with CML and healthy donors. CD1a+CD14- DCs were generated from CD34-/low HPSCs and evaluated morphologically and functionally. CD34+ cells are frequently selected for transplantation and the entire CD34-/low HPSC fraction is wasted. Here, we anticipated the CD34- HPSC subset to constitute an invaluable source for acquiring DCs for immunotherapy. CD34+ and CD34- HPSCs were sorted from the bone marrow samples of CML patients and healthy donors and differentiated ex vivo in a similar way. DCs from CD34-Lin- and CD34+Lin- HPSCs expressed comparable surface markers (CD80, CD83, CD86, HLA-DR, CD40, and CD54). Functional analysis revealed that DCs acquired from both subsets retained a potent allogeneic T cell stimulatory capacity and an efficient phagocytic ability and showed a similar BCR/ABL translocation status. In conclusion, DCs were successfully differentiated from the CD34-Lin- cell subset and showed potent functional capacities, indicating their potential for application in immunotherapy and basic research.

Single nucleotide polymorphisms in matrix metalloproteinase 2 (MMP2) enhance BRAFV600E mutation-mediated oncogenicity and invasiveness of papillary thyroid cancer cells.

Thyroid cancer is a common endocrine neoplasm. Despite its good prognosis, it can lead to significant morbidity and mortality due to metastasis and recurrence. However, the factors involved in metastasis are not well studied. Therefore, we selected matrix metalloproteinases 2 (MMP2) and determined whether it has any role in thyroid cancer. We sequenced the exons of MMP2 in 211 samples including 16 multi-nodular goiters and 195 differentiated thyroid cancers. We identified four non-synonymous single nucleotide polymorphisms (SNPs) of the MMP2 gene in 3.06% (6/195) thyroid cancers. Of the four tumors harboring MMP2 SNPs, three (75%) concomitantly had BRAFV600E. Hence, we speculated that the MMP2 SNPs may cooperate with BRAFV600E in promoting tumor aggressiveness. Overexpression of two MMP2 SNPs (P38L and T458I) exhibited markedly enhanced gelatinase activity with an intact dimerization and induced strong cortactin foci formation in HEK293T cells. Stable expression of the two MMP2 SNPs in BRAFV600E positive BCPAP cells dramatically enhanced cell proliferation, colony formation, and focus formation. Analysis of the morphology of MMP2 SNP bearing BCPAPV600E cells exhibited highly invasive phenotypes characterized by a high rate of wound healing and enhanced cell invasion compared with parental BCPAPV600E cells bearing vector. We also determined that BCPAPV600E cells stably transfected with MMP2 SNPs were highly sensitive to the treatment of BRAF inhibitor, PLX4720. Our study demonstrates that MMP2 SNPs could cooperate with BRAFV600E to promote oncogenicity, migration, and invasiveness of PTC cells. These results suggest that a subset of papillary thyroid cancer with this genetic makeup may benefit from BRAF-mediated therapeutic interventions.

Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers.

BACKGROUND: Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts. METHODS: Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro. RESULTS: We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC. CONCLUSION: Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance.

PAC, a novel curcumin analogue, has anti-breast cancer properties with higher efficiency on ER-negative cells.

We have investigated here the anti-breast cancer properties of two novel curcumin analogues, EAC and PAC. Apoptosis was assessed by the annexin V/propidium iodide (PI) assay on different breast cancer and normal cells. Immunoblotting analysis determined the effects of these agents on different apoptotic and oncogenic proteins. Furthermore, flow cytometry and Elispot were utilised to investigate the effects on the cell cycle and the production of cytokines, respectively. Breast cancer tumour xenografts were developed in nude mice. Finally, (18)F-radiolabeled PAC and curcumin were produced to study their bioavailability and tissue biodistribution in mice. PAC is five times more efficient than curcumin and EAC in inducing apoptosis, mainly via the internal mitochondrial route. This effect was 10-fold higher against ER-negative as compared to ER-positive cells, and ectopic expression of ERα rendered ER-negative breast cancer cells more resistant to PAC. In addition, PAC delayed the cell cycle at G2/M phase with a stronger effect on ER-negative cells. Moreover, PAC exhibited strong capacity as an immuno-inducer through reducing the secretion of the two major Th2 cytokines IL-4 and IL-10. Importantly, PAC significantly reduced tumour size, and triggered apoptosis in vivo. Furthermore, PAC inhibited survivin, NF-kB and its downstream effectors cyclin D1 and Bcl-2, and strongly up-regulated p21(WAF1) both in vitro and in tumours. Besides, PAC exhibited higher stability in blood and greater biodistribution and bioavailability than curcumin in mice. These results indicate that PAC could constitute a powerful, yet not toxic, new chemotherapeutic agent against ER-negative breast tumours.

Defective gammadelta T-cell function and granzyme B gene polymorphism in a cohort of newly diagnosed breast cancer patients.

OBJECTIVE: The purpose of this study was to examine the antitumor immune function of gammadelta T cells and to scan the granzyme B gene for the known single nucleotide polymorphism in breast cancer patients and normal controls. MATERIALS AND METHODS: Levels, cytotoxicity, and functional capacity of gammadelta T cells in peripheral blood mononuclear cells were assessed by flow cytometry, (51)Cr release, and ELISpot assays, respectively. Furthermore, sequence based typing was adopted to screen for granzyme B gene polymorphism. RESULTS: We have found that the frequency and function of gammadelta T cells are reduced both in peripheral blood mononuclear cells of 30 newly diagnosed breast cancer patients (2 [1.2, 3]), compared with 38 normal controls (3.2 [2.5, 5.7]) (p=0.02). In addition, resting gammadelta T cells from breast cancer patients produced significantly more interleukin-6 and tumor necrosis factor-alpha than normal controls. Moreover, ex vivo stimulation of gammadelta T cells with zoledronic acid and interleukin-2 compensated in part for this deficiency, as it stimulated the proliferation, cytokine production, and enhanced the expression of messenger RNA of granzyme B. Interestingly, when the known granzyme B gene polymorphism was screened, we found the prevalence of the mutated genotype RAH/RAH to be significantly (p<0.017) associated with breast cancer patients (14.30%) compared with normal donors (1.40%). Cytotoxicity exerted by gammadelta T cells on Daudi and MCF-7 was significantly higher in donors with the wild-type QPY/QPY (50%) compared with donors with RAH/RAH (21%). CONCLUSIONS: Our data suggest that reduction in the proportion of gammadelta T cells and granzyme B gene polymorphism leads to defective immune function in breast cancer patients. Treatment with zoledronic acid amend partially this fault. Further studies of gammadelta T cells function and granzyme B gene polymorphism in cancers, as well as the potential therapeutic use of zoledronic acid are warranted.

Vincristine and lomustine induce apoptosis and p21(WAF1) up-regulation in medulloblastoma and normal human epithelial and fibroblast cells.

Medulloblastomas arise in the cerebellum and are the most common pediatric primary malignant brain tumors. Currently, medulloblastoma patients are best treated with surgical removal of the tumor, adjuvant radiation therapy and chemotherapy. The chemotherapeutic agents that showed efficiency against medulloblastomas include lomustine and vincristine. However, the effects of these drugs on medulloblastomas as well as on other cell types is still not well defined. In the present report we present evidence that the cytotoxic effect of these drugs is not specific for medulloblastoma cells but includes also normal fibroblast and epithelial cells. We have also shown that vincristine and lomustine trigger apoptosis in all these cells through the mitochondrial pathway via decrease in the level of the anti-apoptosis proteins Bcl-2 and Bcl-xl, respectively. Intriguingly, the proportion of apoptotic cells induced in medulloblastoma and normal epithelial and fibroblastic cells was similar. In addition, vincristine induced low proportion of necrosis in medulloblastoma and normal fibroblast cells. Interestingly, while vincristine induced cell cycle delay in G2/M phase in normal as well as medulloblastoma cells, lomustine effect on the cell cycle was specific for medulloblastoma cells. Furthermore, we have shown that vincristine and lomustine up-regulated p21 protein level in a p53-independent manner. These results shed more light on the biological effects of vincristine and lomustine and show that lomustine is a more specific and potent anti-medulloblastoma agent.

Role of phosphatidylinositol 3'-kinase/AKT pathway in diffuse large B-cell lymphoma survival.

Phosphatidylinositol 3'-kinase (PI3K) is a key player in cell-growth signaling in a number of lymphoid malignancies, but its role in diffuse large B-cell lymphoma (DLBCL) has not been fully elucidated. Therefore, we investigated the role of the PI3K/AKT pathway in a panel of 5 DLBCL cell lines and 100 clinical samples. Inhibition of PI3K by a specific inhibitor, LY294002, induced apoptosis in SUDHL4, SUDHL5, and SUDHL10 (LY-sensitive) cells, whereas SUDHL8 and OCI-LY19 (LY-resistant) cells were refractory to LY294002-induced apoptosis. AKT was phosphorylated in 5 of 5 DLBCL cell lines and inhibition of PI3K caused dephosphorylation/inactivation of constitutively active AKT, FOXO transcription factor, and GSK3 in LY-sensitive cell lines. In addition, there was a decrease in the expression level of inhibitory apoptotic protein, XIAP, in the DLBCL cell lines sensitive to LY294002 after treatment. However, no effect was observed in XIAP protein levels in the resistant DLBCL cell lines following LY294002 treatment. Finally, using immunohistochemistry, p-AKT was detected in 52% of DLBCL tumors tested. Furthermore, in univariate analysis, high p-AKT expression was associated with short survival. In multivariate analysis, this correlation was no longer significant. Altogether, these results suggest that the PI3K/AKT pathway may be a potential target for therapeutic intervention in DLBCL.

Curcumin suppresses growth and induces apoptosis in primary effusion lymphoma.

The mechanisms that regulate induction of the antiapoptotic state and mitogenic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we found that curcumin (diferuloylmethane), a natural compound isolated from the plant Curcuma Ionga, inhibits cell proliferation and induces apoptosis in a dose dependent manner in several PEL cell lines. Such effects of curcumin appear to result from suppression of the constitutively active STAT3 through inhibition of Janus kinase 1 (JAK1). Our data also demonstrate that curcumin induces loss of mitochondrial membrane potential with subsequent release of cytochrome c and activation of caspase-3, followed by polyadenosin-5'-diphosphate-ribose polymerase (PARP) cleavage. Altogether, our findings suggest a novel function for curcumin, acting as a suppressor of JAK-1 and STAT3 activation in PEL cells, leading to inhibition of proliferation and induction of caspase-dependent apoptosis. Therefore, curcumin may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of STAT3.

Inhibition of phosphatidylinositol 3'-kinase/AKT signaling promotes apoptosis of primary effusion lymphoma cells.

PURPOSE: Phosphatidylinositol 3'-kinase (PI3'-kinase) can be activated by the K1 protein of Kaposi sarcoma-associated herpes virus (KSHV). However, the role of PI3'-kinase in KSHV-associated primary effusion lymphoma (PEL) is not known. To assess this, we studied survival and apoptosis in PEL cell lines following inhibition of PI3'-kinase. EXPERIMENTAL DESIGN: Constitutive activation of several targets of PI3-kinase and apoptotic proteins were determined by Western blot analysis using specific antibodies. We used LY294002 to block PI3'-kinase/AKT activation and assess apoptosis by flow cytometric analysis. RESULTS: Blocking PI3'-kinase induced apoptosis in PEL cells, including BC1, BC3, BCBL1, and HBL6, whereas BCP1 was refractory to LY294002-induced apoptosis. LY294002-induced apoptosis did not seem to involve Fas/Fas-L but had an additive effect to CH11-mediated apoptosis. We also show that AKT/PKB is constitutively activated in all PELs and treatment with LY294002 causes complete dephosphorylation in all cell lines except BCP1 where a residual AKT phosphorylation remained after 24 hours of treatment. FKHR and GSK3 were also constitutively phosphorylated in PELs and treatment with LY294002 caused their dephosphorylation. Although inhibition of PI3'-kinase induced cleavage of BID in all cell lines, cytochrome c was released from the mitochondria and caspase-9 and caspase-3 were activated in LY294002-induced apoptotic BC1 but not in resistant BCP1. Similarly, XIAP, a target of AKT, was down-regulated after LY294002 treatment only in sensitive PEL cells. CONCLUSIONS: Our data show that the PI3'-kinase pathway plays a major role in survival of PEL cells and suggest that this cascade may be a promising target for therapeutic intervention in primary effusion lymphomas.

The tumor suppressor p16(INK4a) gene is a regulator of apoptosis induced by ultraviolet light and cisplatin.

p16(INK4a) (hereafter referred to as p16), a major cyclin-dependent kinase (CDK) inhibitor, is the product of a tumor-suppressor gene that has been found inactivated in different cancer types. In the present study, we sought to investigate the role of p16 in apoptosis induced by ultraviolet light (the most important etiological cause of skin cancer) and cisplatin (an anticancer DNA damaging agent). It is clearly shown that p16-compromised osteosarcoma U2OS cell line and p16-/- mouse embryo fibroblasts are sensitive to UV-induced apoptosis, as compared to their respective isogenic p16-expressing cells (EH1, EH2) and p16 +/+, indicating that p16 protects cells from undergoing apoptosis in response to UV light. Importantly, this reduction in UV-mediated apoptosis was associated with downregulation of the proapoptotic Bax protein, with no effect on Bcl-2 expression, suggesting that this antiapoptotic role of p16 is mediated via the intrinsic-mitochondrial pathway. On the other hand, p16 sensitized cells to cisplatin-mediated apoptosis through Bcl-2 decline. Interestingly, only proliferating but not G1-arrested EH1 cells underwent apoptosis in response to the anticancer drug. These novel findings provide further insight into the role of p16 in carcinogenesis, and has potential implications for future therapy strategies.