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Lysosomal enzymes function optimally at low pH; as accumulation of waste material contributes to cell aging and disease, dysregulation of lysosomal pH may represent an early step in several pathologies. Here, we demonstrate that stimulation of the P2X7 receptor (P2X7R) for ATP alkalinizes lysosomes in cultured human retinal pigmented epithelial (RPE) cells and impairs lysosomal function. P2X7R stimulation did not kill RPE cells but alkalinized lysosomes by 0.3 U. Receptor stimulation also elevated cytoplasmic Ca2+; Ca2+ influx was necessary but not sufficient for lysosomal alkalinization. P2X7R stimulation decreased access to the active site of cathepsin D. Interestingly, lysosomal alkalinization was accompanied by a rise in lipid oxidation that was prevented by P2X7R antagonism. Likewise, the autofluorescence of phagocytosed photoreceptor outer segments increased by lysosomal alkalinization was restored 73% by a P2X7R antagonist. Together, this suggests that endogenous autostimulation of the P2X7R may oxidize lipids and impede clearance. The P2X7R was expressed on apical and basolateral membranes of mouse RPE; mRNA expression of P2X7R and extracellular ATP marker NTPDase1 was raised in RPE tissue from the ABCA4−/− mouse model of Stargardt's retinal degeneration. In summary, P2X7R stimulation raises lysosomal pH and impedes lysosomal function, suggesting a possible role for overstimulation in diseases of accumulation.—Guha, S., Baltazar G. C., Coffey, E. E., Tu, L.-A., Lim, J. C., Beckel, J. M., Patel, S., Eysteinsson, T., Lu, W., O'Brien-Jenkins, A., Laties, A. M., Mitchell, C. H. Lysosomal alkalinization, lipid oxidation, and reduced phagosome clearance triggered by activation of the P2X7 receptor.

Purpose: The phosphatidylinositol 3′-kinase (PI3K) family plays a key regulatory role in various cancer-associated signal transduction pathways. Here, we investigated the genomic alterations and gene expression of most known PI3K family members in human epithelial ovarian cancer. Experimental Design: The DNA copy number of PI3K family genes was screened by a high-resolution array comparative genomic hybridization in 89 human ovarian cancer specimens. The mRNA expression level of PI3K genes was analyzed by microarray retrieval approach, and further validated by real-time reverse transcription-PCR. The expression of p55γ protein in ovarian cancer was analyzed on tissue arrays. Small interfering RNA was used to study the function of PIK3R3 in ovarian cancer. Results: In ovarian cancer, 6 of 12 PI3K genes exhibited significant DNA copy number gains (>20%), including PIK3CA (23.6%), PIK3CB (27.0%), PIK3CG (25.8%), PIK3R2 (29.2%), PIK3R3 (21.3%), and PIK3C2B (40.4%). Among those, only PIK3R3 had significantly up-regulated mRNA expression level in ovarian cancer compared with normal ovary. Up-regulated PIK3R3 mRNA expression was also observed in liver, prostate, and breast cancers. The PIK3R3 mRNA expression level was significantly higher in ovarian cancer cell lines (n = 18) than in human ovarian surface epithelial cells (n = 6, P = 0.002). Overexpression of p55γ protein in ovarian cancer was confirmed by tissue array analysis. In addition, we found that knockdown of PIK3R3 expression by small interfering RNA significantly increased the apoptosis in cultured ovarian cancer cell lines. Conclusion: We propose that PIK3R3 may serve as a potential therapeutic target in human ovarian cancer.

Expression profiling using microarrays has become an essential tool for interrogating tumor biology. However, profiling of whole tumor RNA reflects both tumor and host cells, making it difficult to dissect molecular events within specific cellular compartments in the tumor microenvironment. We developed and optimized a simple, rapid technique combining immunohistochemistry and laser-capture microdissection (immuno-LCM) to purify specific cell populations from the tumor microenvironment followed by RNA isolation and amplification for microarray analysis. Using this methodology, we were able to elucidate the in situ expression profile of pure tumor cells and tumor endothelial cells from ovarian tumors with brisk immune infiltrates. This technique not only increased the specificity of profiling isolated cell populations, eliminating genes expressed by surrounding cells, but also increased the sensitivity of analysis, allowing for the detection of low expression genes that were not detected in whole tumor arrays. Pathway analysis of tumor cells in situ identified distinct activation of signaling pathways converging on NF-kappaB, as compared to pathways identified in cultured tumor cell lines, which were primarily metabolic. Profiling of tumor vascular cells revealed most known panendothelial and tumor endothelial-specific markers, and unveiled genes specific to the myeloid-monocytic lineage. We propose that immuno-LCM coupled with transcriptional profiling is a convenient tool for dissecting molecular and cellular events in complex biological systems such as the tumor microenvironment.

The protein kinase C (PKC) family plays a key regulatory role in a wide range of cellular functions as well as in various cancer-associated signal transduction pathways. Here, we investigated the genomic alteration and gene expression of most known PKC family members in human ovarian cancer. The DNA copy number of PKC family genes was screened by a high-resolution array-based comparative genomic hybridization in 89 human ovarian cancer specimens. Five PKC genes exhibited significant DNA copy number gains, including PKCι (43.8%), PKCβ1 (37.1%), PKCγ (27.6%), PKCζ (22.5%), and PKC𝛉 (21.3%). None of the PKC genes exhibited copy number loss. The mRNA expression level of PKC genes was analyzed by microarray retrieval approach. Two of the amplified PKC genes, PKCι and PKC𝛉, were significantly up-regulated in ovarian cancer compared with normal ovary. Increased PKCι expression correlated with tumor stage or grade, and PKCι overexpression was seen mostly in ovarian carcinoma but not in other solid tumors. The above results were further validated by real-time reverse transcription-PCR with 54 ovarian cancer specimens and 24 cell lines; overexpression of PKCι protein was also confirmed by tissue array and Western blot. Interestingly, overexpressed PKCι did not affect ovarian cancer cell proliferation or apoptosis in vitro. However, decreased PKCι expression significantly reduced anchorage-independent growth of ovarian cancer cells, whereas overexpression of PKCι contributed to murine ovarian surface epithelium transformation in cooperation with mutant Ras. We propose that PKCι may serve as an oncogene and a biomarker of aggressive disease in human ovarian cancer. (Cancer Res 2006; 66(9): 4627-35)

Epithelial ovarian cancer is the most frequent cause of gynecologic malignancy-related mortality in women. To identify genes up-regulated in ovarian cancer, PCR-select cDNA subtraction was done and Drosophila Eyes Absent Homologue 2 (EYA2) was isolated as a promising candidate. The transcriptional coactivator eya controls essential cellular functions during organogenesis of Drosophila. EYA2 mRNA was found to be up-regulated in ovarian cancer by real-time reverse transcription–PCR, whereas its protein product was detected in 93.6% of ovarian cancer specimens by immunohistochemistry (n = 140). EYA2 was amplified in 14.8% of ovarian carcinomas, as detected by array-based comparative genomic hybridization (n = 88). Most importantly, EYA2 overexpression was significantly associated with short overall survival in advanced ovarian cancer (n = 99, P = 0.0361). EYA2 was found to function as transcriptional activator in ovarian cancer cells by Gal4 assay and to promote tumor growth in vivo in xenograft models. Therefore, this study suggests an important role of EYA2 in ovarian cancer and its potential application as a therapeutic target.

Phosphatidylinositol 3-kinase (PI3-kinase) is a novel intracellular transducer involved in a wide range of cancer-associated signaling pathways, which comprises various isoforms and splice variants with distinct biologic activities and clinical implications. Especially, the class Ia PI3-kinase 110 kD catalytic subunit alpha (PIK3CA) is the most important isoform in tumorigenesis and possibly, tumor angiogenesis. Several strategies have been developed to block PI3-kinase for cancer therapy; however, the approach to target specific PI3-kinase isoform has not been explored to date. In the present study, we show that RNA interference (RNAi) through small interfering (siRNA) sequences targeting PIK3CA has potential applications in isoform-specific "knock-down" of PI3-kinase. This strategy provides a novel tool to study the function of various PI3-kinase isoforms and may contribute to isoform-specific targeting of PI3-kinase in human cancer.

Tumor growth results in hypoxia. Understanding the mechanisms of gene expression reprogramming under hypoxia may provide important clues to cancer pathogenesis. We studied miRNA genes that are regulated by hypoxia in ovarian cancer cell lines by TaqMan miRNA assay containing 157 mature miRNAs. MiR-210 was the most prominent miRNA consistently stimulated under hypoxic conditions. We provide evidence for the involvement of the HIF signaling pathway in miR-210 regulation. Biocomputational analysis and in vitro assays demonstrated that e2f transcription factor 3 (e2f3), a key protein in cell cycle, is regulated by miR-210. E2F3 was further confirmed to be downregulated at the protein level upon induction of miR-210. Importantly, we found remarkably high frequency of miR-210 gene copy deletions in ovarian cancer patients (64%, n = 114) and that gene copy number correlates with miR-210 expression levels. Taken together, our results indicate that miR-210 plays a crucial role in tumor onset as a key regulator of the hypoxia response and provide evidence for a link between hypoxia and the regulation of cell cycle.

MicroRNAs (miRNAs) are endogenous noncoding RNAs, which negatively regulate gene expression. To determine genomewide miRNA DNA copy number abnormalities in cancer, 283 known human miRNA genes were analyzed by high-resolution array-based comparative genomic hybridization in 227 human ovarian cancer, breast cancer, and melanoma specimens. A high proportion of genomic loci containing miRNA genes exhibited DNA copy number alterations in ovarian cancer (37.1%), breast cancer (72.8%), and melanoma (85.9%), where copy number alterations observed in >15% tumors were considered significant for each miRNA gene. We identified 41 miRNA genes with gene copy number changes that were shared among the three cancer types (26 with gains and 15 with losses) as well as miRNA genes with copy number changes that were unique to each tumor type. Importantly, we show that miRNA copy changes correlate with miRNA expression. Finally, we identified high frequency copy number abnormalities of Dicer1 , Argonaute2 , and other miRNA-associated genes in breast and ovarian cancer as well as melanoma. These findings support the notion that copy number alterations of miRNAs and their regulatory genes are highly prevalent in cancer and may account partly for the frequent miRNA gene deregulation reported in several tumor types.

Vascular remodeling in host tissues surrounding growing tumors is implicated in the successful development of tumor neovasculature. Cooperation between vascular endothelial growth factor (VEGF) and angiopoietins (Angs) is considered to be critical in this context. However, the mechanisms regulating the coordinated expression of these molecules remain, to date, elusive. In this study, we used a murine ovarian cancer angiogenesis model induced by overexpression of VEGF, as well as 52 human ovarian cancer specimens and 36 established cancer cell lines to characterize the expression and regulation of Ang-2 in the context of tumor angiogenesis. Using a combination of immunohistochemistry, laser capture microdissection and real-time quantitative reverse transcription-PCR, we showed that tumor-derived VEGF significantly up-regulated the expression of Ang-2 in host stroma endothelial cells, resulting in markedly increased Ang-2/Tie-2 mRNA copy number ratio in vivo. In vitro experiments showed that VEGF directly up-regulated Ang-2, which is mediated via VEGF receptor-2/flk-1/KDR pathway, in cultured endothelial cells through transcriptional activation rather than the enhanced mRNA stability. In human ovarian cancer, Ang-2 was primarily expressed in stroma endothelial cells and detectable in tumor cells of only 12% tumor specimens; however, it was not detected in the majority of established ovarian cancer cell lines. In addition, a significant correlation was observed between VEGF and Ang-2 mRNA expression (P0.01) but not between VEGF and Ang-1 or Tie-2 in human ovarian cancer specimens. In the mouse ovarian cancer model, up-regulation of Ang-2 in host stroma endothelial cells was significantly associated with pericyte loss and instability of the host vasculature surrounding the tumor. Our study suggests a novel mechanism by which tumor-derived VEGF interacts with Angs/Tie-2 system in host stroma endothelial cells and induces in a paracrine manner the remodeling of host vasculature to support angiogenesis during tumor growth.

We have previously shown that in the dopamine-rich anterior cingulate cortex (ACC), significant changes in gamma-aminobutyric acid (GABA) immunoreactivity occur in the offspring of rabbits given intravenous injections of cocaine (3 mg/kg) twice daily during pregnancy. In the present study, the effects of prenatal cocaine exposure on the developmental expression of specific GABA(A) receptor subunit mRNAs were investigated. We compared the distribution of the alpha1, beta2, and gamma2 subunit mRNAs in cocaine- and saline-treated offspring aged postnatal days 20 and 60 (P20, P60). At P20, prenatal cocaine exposure resulted in a significant increase in alpha1 subunit mRNA in ACC lamina III and a significant reduction in the amounts of the beta2 subunit mRNA in ACC lamina II. No differences between cocaine- and saline-treated controls were detected for gamma2 subunit mRNA levels in ACC. Although the pattern of labeling was altered in cocaine-exposed animals, Nissl sections revealed no differences in lamination, indicating that the changes in GABA(A) subunit mRNAs could not be attributed to abnormal cytoarchitectonics. In P60 brains, no significant differences were observed between cocaine- and saline-treated material, indicating that the observed differences were transient. Collectively, our data show that prenatal cocaine exposure elicits differential, lamina-specific changes in mRNA levels encoding selected subunits of the GABA(A) receptor. Since these changes occur during a critical period when fine tuning of synaptic organization is achieved by processes of selective elimination or stabilization of synapses, we suggest that specific subunit mRNAs of the GABA(A) receptor play a role in cortical development.

Galactokinase (GK; EC 2.7.1.6) is the first enzyme in the metabolism of galactose. In humans, GK deficiency results in congenital cataracts due to an accumulation of galactitol within the lens. In an attempt to make a galactosemic animal model, we cloned the mouse GK gene (Glk1) and disrupted it by gene targeting. As expected, galactose was very poorly metabolized in GK-deficient mice. In addition, both galactose and galactitol accumulated in tissues of GK-deficient mice. Surprisingly, the GK-deficient animals did not form cataracts even when fed a high galactose diet. However, the introduction of a human aldose reductase transgene into a GK-deficient background resulted in cataract formation within the first postnatal day. This mouse represents the first mouse model for congenital galactosemic cataract.

Previous studies of rabbits exposed in utero to cocaine have revealed an increase in the number of neurons which are GABA immunoreactive in the anterior cingulate cortex (ACC), suggesting a cocaine-elicited modification in the balance of excitatory and inhibitory interactions. Of the major calcium binding proteins expressed by different subgroups of GABAergic neurons, parvalbumin has been observed in conditions involving excess excitation, and may serve to protect neurons from excitotoxicity. In the present study, we used immunocy-tochemistry to compare the effects of prenatal cocaine exposure on the postnatal development of parvalbumin immunoreactivity in interneurons of the visual cortex (VC) and ACC. We determined the number and laminar distribution of parvalbumin immunoreactive neurons, and we also assessed the distribution of parvalbumin immunoreactivity within primary, secondary and tertiary dendrites of neurons in these two cortical areas. In both ACC and VC, parvalbumin immunoreactive neurons were first observed around postnatal day 10 (P10) and their number increased rapidly from P10 to P20. At all ages studied (P10 to P60) there was no difference between cocaine-exposed and saline control animals in the number or laminar distribution of parvalbumin immunoreactive neurons in either cortical area. However, the distribution of parvalbumin immunoreactivity within dendrites revealed a significant difference between cocaine-exposed and saline control animals in ACC but not in VC. In ACC, at all ages studied, there was an increase in the number of primary, secondary and tertiary dendrites which were parvalbumin immunoreactive in cocaine-exposed animals compared with saline controls. This difference was most striking in secondary dendrites, and in laminae V and VI. The effect was observed at doses of 4 and 3 mg/kg per injection but not at 2 mg/kg per injection. In contrast to ACC, in VC there was no difference in the number of immunoreactive dendrites in cocaine-exposed animals compared with saline controls. These observations are consistent with the hypothesis that the ACC of rabbits exposed in utero to cocaine is characterized by altered excitatory/inhibitory interactions. ACC receives a dense dopaminergic input, but VC receives minimal dopaminergic innervation. Mechanisms by which the action of cocaine on the developing dopaminergic system may modify the balance of excitation and inhibition in ACC are discussed.

Anterior cingulate cortex develops abnormally in rabbits exposed to cocaine in utero but visual cortex is normal. The interactions of cocaine with the dopamine, norepinephrine and serotonin systems makes each a potential candidate for influencing these developmental effects. Here, we report no differences in the distribution and density of serotonin and tyrosine hydroxylase-immunoreactive fibers in the anterior cingulate cortex in postnatal rabbits exposed prenatally to cocaine or saline. Because the pattern and extent of cortical innervation by these systems appears normal, the data suggest that the effects of prenatal exposure to cocaine may involve changes in monoamine signaling distinct from morphological reorganization of the cortical afferents.