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Tissue microarray technology facilitates rapid assessment of expression of molecular markers by enabling the simultaneous analysis of hundreds of tissue specimens. We have applied this technology to establish a microarray composed of cell pellets derived from 40 human lymphoma/leukemiaderived cell lines harboring a variety of molecular abnormalities. The application of cell line microarrays to the assessment of biologic marker evaluation was validated by studying the immunohistochemical expression of PTEN and phosphorylated AKT, two mediators of the phosphatidylinositol (PI)-3-kinase pathway. In addition to the high throughout analysis of protein expression in lymphoma/leukemia cells, this methodology also enables the evaluation of subcellular localization of protein expression. Cytoplasmic PTEN expression was observed in the majority of cell lines (87%), whereas a minor subset demonstrated nuclear expression. Phosphorylated AKT was also expressed predominantly within the cytoplasm in 65% of cell lines, whereas nuclear expression was seen in a minority. An inverse relationship between PTEN and phosphorylated AKT was observed in 63% of cell lines. No cell lines showed absence of PTEN expression, whereas 50% of cell lines showed low PTEN expression. Our data support the integrity of the PI-3-kinase-PTEN-AKT pathway in a majority of cell lines derived from hematologic malignancies and clearly demonstrates the utility of microarray technology in the in situ assessment of expression of molecular markers in tumor-derived cell lines.

Overexpression of Bcl-2 protein occurs via both t (14;18)-dependent and independent mechanisms and contributes to the survival and chemoresistance of nonHodgkin lymphomas. HA14-1 is a nonpeptidic organic small molecule, which has been shown to inhibit the interaction of Bcl-2 with Bax, thereby interfering with the antiapoptotic function of Bcl-2. In this study, we sought to determine the in vitro efficacy of HA14-1 as a therapeutic agent for non-Hodgkin lymphomas expressing Bcl-2. Assessment of cell viability demonstrated that HA14-1 induced a dose-(IC 50 =10 μM) and time-dependent growth inhibition of a cell line (SudHL-4) derived from a t(14;18)-positive, Bcl-2-positive, non-Hodgkin lymphoma. HA14-1 effectively induced apoptosis via a caspase 3-mediated pathway but did not affect either the p38 MAPK or p44/42 MAPK pathways. Western blot analyses of Bcl-2 family proteins and other cell cycle-associated proteins were performed to determine the molecular sequelae of HA14-1-induced apoptosis. The results show down-regulation of Mcl-1 but up-regulation of p27 kip1 , Bad, Bcl-xL, and Bcl-2 proteins, without change in Bax levels during HA14-1-mediated apoptosis. Our findings further elucidate the cellular mechanisms accompanying Bcl-2 inhibition and demonstrate the potential of Bcl-2 inhibitors as therapeutic agents for the treatment of non-Hodgkin lymphomas.

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