Citations

ABSTRACT Neurofibrillary tangles (NFTs) are a characteristic neuropathological feature of Alzheimer's disease (AD), and molecular chaperones appear to be involved in the removal of disease‐associated hyperphosphorylated tau, a primary component of NFTs. Here, novel HSP90 inhibitors were used to examine the impact of chaperone elevation on clearance of different tau species in transfected cells using a unique quantitative assay. The HSP90 inhibitors reduced levels of tau phosphorylated at proline‐directed Ser/Thr sites (pS202/T205, pS396/S404) and conformationally altered (MC‐1) tau species, an epitope that is immeasurable by standard Western blot techniques. The selective clearance of these phospho‐tau species and MC‐1 tau was mediated via the proteasome, while lysosomal‐mediated tau degradation seems to lack specificity for certain tau species, suggesting a more general role in total tau removal. Interestingly, tau phosphorylated at S262/S356 within the tau microtubule binding domain was minimally affected by chaperone induction. Overall, our data show that chaperone induction results in the selective clearance of specific phospho‐tau and conformationally altered tau species mediated by the proteasome; however, the apparent stability of pS262/S356 tau may also explain why MARK is able to regulate normal tau function yet still be linked to the initiation of pathogenic tau hyperphosphorylation in AD.

AbstractCREB-binding protein (CBP) and its para-log p300 are transcriptional coactivators that physically or functionally interact with over 320 mammalian and viral proteins, including 36 that are essential for B cells in mice. CBP and p300 are generally considered limiting for transcription, yet their roles in adult cell lineages are largely unknown since homozygous null mutations in either gene or compound heterozygosity cause early embryonic lethality in mice. We tested the hypotheses that CBP and p300 are limiting and that each has unique properties in B cells, by using mice with Cre/LoxP conditional knockout alleles for CBP (CBPflox) and p300 (p300flox), which carry CD19Cre that initiates floxed gene recombination at the pro–B-cell stage. CD19Cre-mediated loss of CBP or p300 led to surprisingly modest deficits in B-cell numbers, whereas inactivation of both genes was not tolerated by peripheral B cells. There was a moderate decrease in B-cell receptor (BCR)–responsive gene expression in CBP or p300 homozygous null B cells, suggesting that CBP and p300 are essential for this signaling pathway that is crucial for B-cell homeostasis. These results indicate that individually CBP and p300 are partially limiting beyond the pro-B-cell stage and that other coactivators in B cells cannot replace their combined loss.

Abstract Aromatase [cytochrome P450 19 (CYP19)] is a critical enzyme for estrogen biosynthesis, and aromatase inhibitors are of increasing importance in the treatment of breast cancer. We set out to identify and characterize genetic polymorphisms in the aromatase gene, CYP19, as a step toward pharmacogenomic studies of aromatase inhibitors. Specifically, we “resequenced” all coding exons, all upstream untranslated exons plus their presumed core promoter regions, all exon-intron splice junctions, and a portion of the 3′-untranslated region of CYP19 using 240 DNA samples from four ethnic groups. Eighty-eight polymorphisms were identified, resulting in 44 haplotypes. Functional genomic studies were done with the four nonsynonymous coding single nucleotide polymorphisms (cSNP) that we observed, two of which were novel. Those cSNPs altered the following amino acids: Trp39Arg, Thr201Met, Arg264Cys, and Met364Thr. The Cys264, Thr364, and double variant Arg39Cys264 allozymes showed significant decreases in levels of activity and immunoreactive protein when compared with the wild-type (WT) enzyme after transient expression in COS-1 cells. A slight decrease in protein level was also observed for the Arg39 allozyme, whereas Met201 displayed no significant changes in either activity or protein level when compared with the WT enzyme. There was also a 4-fold increase in apparent Km value for Thr364 with androstenedione as substrate. Of the recombinant allozymes, only the double mutant (Arg39Cys264) displayed a significant change from the WT enzyme in inhibitor constant for the aromatase inhibitors exemestane and letrozole. These observations indicate that genetic variation in CYP19 might contribute to variation in the pathophysiology of estrogen-dependent disease.

Abstract Purpose: The overall 5-year survival of patients with pancreatic cancer remains <5%. Novel therapeutic strategies are needed. We examined the effect of rapamycin, alone and in combination with antiangiogenesis therapy, on pancreatic cancer in vivo. Experimental Design: Human pancreatic cancer AsPC-1 cells were orthotopically injected into severe combined immunodeficient/beige mice to evaluate primary tumor growth and liver metastasis after treatment with rapamycin alone or in combination with anti-vascular endothelial growth factor antibody 2C3. Tumor cell proliferation was determined by bromodeoxyuridine incorporation. To detect tumor cell apoptosis, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used. Tumor angiogenesis was investigated by using a monoclonal anti-CD31 antibody. All statistical tests were two-sided. Results: Rapamycin, alone and in combination with 2C3, strongly inhibited primary and metastatic tumor growth in an orthotopic pancreatic cancer animal model. Furthermore, the combination therapy significantly improved the effect on liver metastasis compared with single treatment with either rapamycin (P = 0.0128) or 2C3 (P = 0.0099). Rapamycin alone inhibited pancreatic tumor cell proliferation, induced apoptosis, and decreased tumor angiogenesis. Nevertheless, the combination therapy showed a significant, stronger inhibition of tumor cell proliferation (P = 0.0002 versus rapamycin alone and P < 0.0001 versus 2C3 alone). The induction of apoptosis was significantly higher than in the rapamycin-treated group (P = 0.0039). Additionally, the combination therapy further improved suppression of tumor cell angiogenesis compared with rapamycin treatment (P = 0.029) Conclusions: Our studies propose new therapeutic strategies to inhibit both primary and metastatic tumor growth in pancreatic cancer. Considering the fact that liver metastasis is a crucial problem in advanced stages of pancreatic cancer, the combination therapy of rapamycin plus anti-vascular endothelial growth factor antibody 2C3 is a significant advantage compared with single treatment with rapamycin.

Abstract Purpose: Advanced-stage epithelial ovarian cancer has a poor prognosis with long-term survival in less than 30% of patients. When the disease is detected in stage I, more than 90% of patients can be cured by conventional therapy. Screening for early-stage disease with individual serum tumor markers, such as CA125, is limited by the fact that no single marker is up-regulated and shed in adequate amounts by all ovarian cancers. Consequently, use of multiple markers in combination might detect a larger fraction of early-stage ovarian cancers. Experimental Design: To identify potential candidates for novel markers, we have used Affymetrix human genome arrays (U95 series) to analyze differences in gene expression of 41,441 known genes and expressed sequence tags between five pools of normal ovarian surface epithelial cells (OSE) and 42 epithelial ovarian cancers of different stages, grades, and histotypes. Recursive descent partition analysis (RDPA) was performed with 102 probe sets representing 86 genes that were up-regulated at least 3-fold in epithelial ovarian cancers when compared with normal OSE. In addition, a panel of 11 genes known to encode potential tumor markers [mucin 1, transmembrane (MUC1), mucin 16 (CA125), mesothelin, WAP four-disulfide core domain 2 (HE4), kallikrein 6, kallikrein 10, matrix metalloproteinase 2, prostasin, osteopontin, tetranectin, and inhibin] were similarly analyzed. Results: The 3-fold up-regulated genes were examined and four genes [Notch homologue 3 (NOTCH3), E2F transcription factor 3 (E2F3), GTPase activating protein (RACGAP1), and hematological and neurological expressed 1 (HN1)] distinguished all tumor samples from normal OSE. The 3-fold up-regulated genes were analyzed using RDPA, and the combination of elevated claudin 3 (CLDN3) and elevated vascular endothelial growth factor (VEGF) distinguished the cancers from normal OSE. The 11 known markers were analyzed using RDPA, and a combination of HE4, CA125, and MUC1 expression could distinguish tumor from normal specimens. Expression at the mRNA level in the candidate markers was examined via semiquantitative reverse transcription-PCR and was found to correlate well with the array data. Immunohistochemistry was performed to identify expression of the genes at the protein level in 158 ovarian cancers of different histotypes. A combination of CLDN3, CA125, and MUC1 stained 157 (99.4%) of 158 cancers, and all of the tumors were detected with a combination of CLDN3, CA125, MUC1, and VEGF. Conclusions: Our data are consistent with the possibility that a limited number of markers in combination might identify >99% of epithelial ovarian cancers despite the heterogeneity of the disease.

AbstractPurpose: The B7 family molecules have been shown to regulate immune responses in both positive and negative fashions. Their roles in the progression of human cancers, however, are not well established. The aim of this study was to examine whether leukemic cells of acute myeloid leukemia express functional B7 family molecules and, if so, whether such expression has any clinical significance.Experimental Design: The expression of four B7 family molecules, B7.1, B7.2, B7-H1, and B7-H2, on leukemic cells from acute myeloid leukemia patients was analyzed by flow cytometry. The function of the expressed molecules was examined by the allogeneic mixed lymphocyte-leukemic cell reaction, and their relationship to the clinical data and survival was analyzed.Results: Although B7.1 and B7-H1 expressions were rare, the cells from a substantial number of acute myeloid leukemia cases expressed B7.2 and B7-H2 molecules [mean percentages of B7.2- and B7-H2-positive cells were 28.9% (n = 58) and 15.3% (n = 59), respectively]. Patients in whom >25% of leukemic cells expressed B7-H2 had significantly shorter survival, and this B7-H2 positivity had the strongest prognostic value when B7-H2 and other prognostic factors were analyzed together by multivariate analysis (P = 0.0108). Furthermore, B7.2 expression was associated with hyperleukocytosis (P = 0.026). Consistent with this finding, acute myeloid leukemia cells expressing B7.2 and B7-H2 induced allogeneic CD4+ T cells to proliferate and secrete interleukin-4 and interleukin-10 in vitro, effects that were partially blocked by antibodies against B7.2 and B7-H2.Conclusions: Our results indicate the expression of functional B7.2 and B7-H2 molecules, and these molecules may facilitate progression of acute myeloid leukemia.

Embryo implantation in the uterus is a critical step in mammalian reproduction, requiring preparation of the uterus receptive to blastocyst implantation. Uterine receptivity, also known as the window of implantation, lasts for a limited period, and it is during this period blastocysts normally implant. Ovarian steroid hormones estrogen and progesterone (P 4 ) are the primary regulators of this process. The immunophilin FKBP52 serves as a cochaperone for steroid hormone nuclear receptors to govern appropriate hormone action in target tissues. Here we show a critical role for FKBP52 in mouse implantation. This immunophilin has unique spatiotemporal expression in the uterus during implantation, and females missing the Fkbp52 gene have complete implantation failure due to lack of attainment of uterine receptivity. The overlapping uterine expression of FKBP52 with nuclear progesterone receptor (PR) in wild-type mice together with reduced P 4 binding to PR, attenuated PR transcriptional activity and down-regulation of several P 4 -regulated genes in uteri of Fkbp52 -/- mice, establishes this cochaperone as a critical regulator of uterine P 4 function. Interestingly, ovulation, another P 4 -mediated event, remains normal. Collectively, the present investigation provides evidence for an in vivo role for this cochaperone in regulating tissue-specific hormone action and its critical role in uterine receptivity for implantation.