Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients.

Bladder cancer (BC) is the 12th most commonly diagnosed cancer worldwide. Although there are several well-established molecular and immunological classifications, prognostic and predictive markers for tumor cells and immune cells are still needed. Using a tissue microarray, we analyzed the expression of the chemokine CC motif ligand 5 (CCL5) by immunohistochemistry (IHC) in 175 muscle-invasive BC samples. The application of a single cutoff for the staining status of tumor cells (TCs; positive vs. negative) and immune cells (ICs; positive vs. negative) revealed 75 patients (42.9%) and 123 patients (70.3%) with CCL5-positive TCs or ICs, respectively. IHC results were associated with prognostic and predictive data. Multivariate Cox regression analysis revealed that positive CCL5 staining in TCs was associated with significantly shorter disease-specific survival (DSS; RR = 1.51; p = 0.047), but CCL5-negative ICs were associated with significantly shorter overall survival (OS; RR = 1.66; p = 0.005), DSS (RR = 2.02; p = 0.001) and recurrence-free survival (RFS; RR = 1.94; p = 0.002). Adjuvant chemotherapy was favorable for patients with CCL5-negative ICs for OS (RR = 0.30; p = 0.006), DSS (RR = 0.36; p = 0.022) and RFS (RR = 0.41; p = 0.046) but not for patients with CCL5-positive ICs, except in the subgroup of N1 + N2 patients, where it was associated with better OS. We suggest that CCL5 expression can be a prognostic and predictive marker for muscle-invasive bladder cancer patients.

Chemerin and Chemokine-like Receptor 1 Are Not Prognostic in Colorectal Carcinoma.

BACKGROUND/AIM: Colorectal carcinoma is a commonly diagnosed malignancy. The chemoattractant protein chemerin was shown to regulate immune response, angiogenesis, and cell migration and has a role in gastrointestinal cancers. Chemerin and its receptor chemokine-like receptor 1 (CMKLR1) are expressed in the colon and here, their diagnostic and prognostic value in colorectal carcinoma was examined. PATIENTS AND METHODS: Chemerin and CMKLR1 protein were quantified by immunohistochemistry in tumor tissues of 86 patients with colorectal cancer. Associations with tumor stage, tumor grade, lymph node score, tumor recurrence, and survival were calculated. RESULTS: Chemerin was not detectable in 12%, low expressed in 55%, medium expressed in 27%, and highly abundant in 7% of the tumors. CMKLR1 protein levels were low in 25%, medium in 70%, and high in 5% of the tumors. Chemerin protein expression was slightly induced in larger tumors of males. CMKLR1 protein expression was modestly higher in the tumors of patients with local and/or distal disease recurrence. CMKLR1 and chemerin protein in colorectal cancer tissues were not related to tumor grade or lymph node score. CD3, CD4, and CD8 protein expression did not correlate with chemerin or CMKLR1. Tumor chemerin and CMKLR1 protein levels were similar in survivors and non-survivors. CONCLUSION: In colorectal carcinoma, chemerin and CMKLR1 proteins are weakly associated with tumor stage and tumor recurrence. Levels of these proteins in colorectal carcinoma tissues are not connected with patients' survival.

Combination of GP88 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Is an Independent Prognostic Factor for Bladder Cancer Patients.

Urothelial bladder cancer (BCa) is the ninth most commonly diagnosed cancer worldwide and accounts for approximately 3% of global cancer diagnoses. We are interested in prognostic markers that may characterize tumor cells (TCs) and immune cells (ICs) and their relationship in BCa. A potential candidate marker that meets these criteria is progranulin (GP88), which is expressed separately in TCs and ICs. We analyzed GP88 expression by immunohistochemistry (IHC) in 196 muscle-invasive BCa samples using a tissue microarray. The immunoreactive score for GP88 staining in TCs and the percentage of GP88-positive ICs was determined. An easy cutoff for the staining status of TCs (positive vs. negative) and ICs (0% vs. >0%) and, more generally, negative vs. positive GP88 staining could be applied. We detected 93 patients (47.4%) and 92 patients (46.9%) with GP88-positive TCs or ICs, respectively. The IHC results were correlated with clinicopathological and survival data. Positive GP88 staining in TCs appeared to be an independent poor prognostic factor for disease-specific survival (DSS) (RR (relative risk) = 1.74; p = 0.009) and recurrence-free survival (RFS) (RR = 1.92; p = 0.002). In contrast, negative GP88 staining in ICs was an independent negative predictor for overall survival (OS) (RR = 2.18; p < 0.001), DSS (RR = 2.84; p < 0.001) and RFS (RR = 2.91; p < 0.001) in multivariate Cox's regression analysis. When combining GP88 staining in TCs and ICs, a specific combination of GP88-positive TCs and GP88-negative ICs was associated with a 2.54-fold increased risk of death, a 4.21-fold increased risk of disease-specific death and a 4.81-fold increased risk of recurrence compared to GP88-negative TCs and GP88-positive ICs. In summary, GP88 positivity in TCs is a negative prognostic factor for DSS and RFS. In addition, GP88 positivity can mark ICs that are associated with a good prognosis (OS, DSS and RFS). The combination of GP88 staining in TCs and ICs appears to be a significant independent prognostic biomarker in muscle-invasive BCa.

Immune Cell-Associated Protein Expression Helps to Predict Survival in Muscle-Invasive Urothelial Bladder Cancer Patients after Radical Cystectomy and Optional Adjuvant Chemotherapy.

Bladder cancer (BCa) is the tenth most commonly diagnosed malignant cancer worldwide. Although adjuvant chemotherapy following radical cystectomy is a common therapy for muscle invasive bladder cancer patients, no applicable biomarkers exist to predict which patients will benefit from chemotherapy. In this study, we examined three immune cell markers, the chemokine CC motif ligand 2 (CCL2), the pan macrophage marker cluster of differentiation 68 (CD68) and the M2 macrophage marker cluster of differentiation 163 (CD163), using immunohistochemistry to determine their predictive value for the chemotherapy response in different nodal stage (pN0 vs. pN1 + 2) and tumor stage subgroups (pT2 vs. pT3 + 4). The prognosis was studied in terms of the overall survival (OS), disease-specific survival (DSS), and recurrence-free-survival (RFS) in 168 muscle invasive BCa patients. Chemotherapy was associated with a poorer prognosis in patients with a higher expression of the immune markers CCL2 (RFS), CD68 (DSS and RFS), and CD163 (DSS and RFS) in the N0 group and with poorer survival in patients with a higher expression of the immune markers CCL2 (OS, DSS, and RFS), CD68 (OS, DSS, and RFS), and CD163 (OS, DSS, and RFS) in the pT2 group when compared with treatments without chemotherapy. In contrast, chemotherapy was associated with a better prognosis in patients with a low expression of the immune markers CCL2 (DSS and RFS), CD68 (OS, DSS, and RFS), and CD163 (OS) in the N1 + 2 group. In addition, chemotherapy was associated with improved survival in patients with a low expression of the immune marker CD68 (OS and DSS) and there was a trend for a better prognosis in patients with a low expression of CD163 (OS) in the pT3 + 4 group compared to patients not treated with chemotherapy. Interestingly, CD68 appeared to be the most applicable immune marker to stratify patients by the outcome of chemotherapy in the nodal stage and tumor stage groups. Overall, we suggest that, in addition to the clinical factors of tumor stage and nodal stage, it is also meaningful to consider the abundance of immune cells, such as macrophages, to better predict the response to chemotherapy for BCa patients after radical treatment.

PIWI-Like 1 and PIWI-Like 2 Expression in Breast Cancer.

PIWI-like 1 and PIWI-like 2 play a role in stem cell self-renewal, and enhanced expression has been reported for several tumor entities. However, few studies have investigated PIWI-like 1 and PIWI-like 2 expressions in breast cancer subtypes regarding prognosis. Therefore, we examined protein expression in a large consecutive cohort of breast cancer patients and correlated it to breast cancer subtypes and survival outcome. PIWI-like 1 and PIWI-like 2 expressions were evaluated using immunohistochemistry in a cohort of 894 breast cancer patients, of whom 363 were eligible for further analysis. Percentage and intensity of stained tumor cells were analyzed and an immunoreactive score (IRS) was calculated. The interaction of PIWI-like 1 and PIWI-like 2 showed a prognostic effect on survival. For the combination of high PIWI-like 1 and low PIWI-like 2 expressions, adjusted hazard ratios (HRs) were significantly higher with regard to overall survival (OS) (HR 2.92; 95% confidence interval (CI) 1.24, 6.90), disease-free survival (DFS) (HR 3.27; 95% CI 1.48, 7.20), and distant disease-free survival (DDFS) (HR 7.64; 95% CI 2.35, 24.82). Both proteins were significantly associated with molecular-like and PAM50 subgroups. Combining high PIWI-like 1 and low PIWI-like 2 expressions predicted poorer prognosis and both markers were associated with aggressive molecular subtypes.

Expression of AR-V7 (Androgen Receptor Variant 7) Protein in Granular Cytoplasmic Structures Is an Independent Prognostic Factor in Prostate Cancer Patients.

Prostate cancer (PCa) is the second most common cancer, causing morbidity and mortality among men world-wide. The expression of the androgen receptor (AR) and its splice variants is a crucial factor of prostate cancer biology that has not been comprehensively studied in PCa tumors. The aim of this study was to characterize the protein expression of the AR and its splice variant, AR-V7, and their subcellular distributions in PCa by immunohistochemistry and to correlate the results to the clinicopathological data and prognosis. Immunohistochemical staining for AR and AR-V7 was performed on a tissue microarray (TMA) with specimens from 410 PCa patients using an immunoreactive score (IRS) or only the percentage of AR-V7 staining in cytoplasmic granules. Nuclear or cytoplasmic AR staining was not associated with prognosis. AR-V7 staining was only occasionally observed in the nucleus. However, AR-V7 staining in the cytoplasm or in cytoplasmic granules was associated with relapse-free survival (RFS). AR-V7 staining of the cytoplasm was associated with a shorter RFS, whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. In a multivariate Cox's regression analysis, only negative (<5%) AR-V7 staining of cytoplasmic granules remained an independent prognostic factor for RFS (HR = 5.3; p = 0.006). In a further subgroup analysis by multivariate Cox's regression analysis, AR-V7 was an independent prognostic factor in the following groups: age ≤ 65 (HR = 9.7; p = 0.029), negative CK20 staining (HR = 7.0; p = 0.008), and positive perineural invasion (HR = 3.7; p = 0.034). Altogether, AR-V7 protein in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.

CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage.

Bladder cancer (BCa) is the ninth most commonly diagnosed cancer worldwide. Although there are several well-established molecular and immunological classifications, markers for tumor cells and immune cells that are associated with prognosis are still needed. The chemokine CC motif ligand 2 (CCL2) could be such a marker. We analyzed the expression of CCL2 by immunohistochemistry (IHC) in 168 muscle invasive BCa samples using a tissue microarray. Application of a single cut-off for the staining status of tumor cells (TCs; positive vs. negative) and immune cells (ICs; ≤6% of ICs vs. >6% of ICs) revealed 57 cases (33.9%) and 70 cases (41.7%) with CCL2-positive TCs or ICs, respectively. IHC results were correlated with clinicopathological and survival data. Positive CCL2 staining in TCs was associated with shorter overall survival (OS), disease-specific survival (DSS), and relapse-free survival (RFS) (p = 0.004, p = 0.036, and p = 0.047; log rank test) and appeared to be an independent prognostic factor for OS (RR = 1.70; p = 0.007; multivariate Cox's regression analysis). In contrast, positive CCL2 staining in the ICs was associated with longer OS, DSS, and RFS (p = 0.032, p = 0.001, and p = 0.001; log rank test) and appeared to be an independent prognostic factor for DSS (RR = 1.77; p = 0.031; multivariate Cox's regression analysis). Most interestingly, after separating the patients according to their lymph node status (N0 vs. N1+2), CCL2 staining in the ICs was differentially associated with prognosis. In the N0 group, CCL2 positivity in the ICs was a positive independent prognostic factor for OS (RR = 1.99; p = 0.014), DSS (RR = 3.17; p = 0.002), and RFS (RR = 3.10; p = 0.002), whereas in the N1+2 group, CCL2 positivity was a negative independent factor for OS (RR = 3.44; p = 0.019)) and RFS (RR = 4.47; p = 0.010; all multivariate Cox's regression analyses). In summary, CCL2 positivity in TCs is a negative prognostic factor for OS, and CCL2 can mark ICs that are differentially associated with prognosis depending on the nodal stage of BCa patients. Therefore, CCL2 staining of TCs and ICs is suggested as a prognostic biomarker for BCa patients.

Exploring the MIR143-UPAR Axis for the Inhibition of Human Prostate Cancer Cells In Vitro and In Vivo.

MIR143 is pathologically downregulated and may function as a tumor suppressor in prostate cancer. Likewise, the urokinase plasminogen activator receptor (UPAR) is overexpressed in prostate carcinoma, representing a negative prognostic marker and putative therapeutic target gene. In this paper, we establish UPAR as a new direct target of MIR143. Luciferase reporter gene constructs identify one of the two in silico-predicted binding sites as functionally relevant for direct MIR143 binding to the 3' UTR, and, concomitantly, transfection of MIR143 reduces UPAR protein levels in prostate carcinoma cells in vitro. Inhibitory effects on cell proliferation and colony formation, spheroid growth and integrity, and cell viability are extensively analyzed, and they are compared to direct small interfering RNA (siRNA)-mediated uPAR knockdown or combined microRNA (miRNA)-siRNA treatment. Switching to a therapeutically more relevant in vivo model, we demonstrate tumor-inhibitory effects of MIR143 replacement therapy by systemic treatment of mice bearing subcutaneous PC-3 tumor xenografts with MIR143 formulated in polymeric nanoparticles. This efficient, nanoparticle-mediated delivery of intact MIR143 mediates the marked downregulation of uPAR protein, but not mRNA levels, thus indicating translational inhibition rather than mRNA degradation. In summary, we identify UPAR as a direct target gene of MIR143, and we establish the therapeutic anti-tumor potential of nanoparticle-based MIR143 replacement in prostate cancer.

Piwi-like 1 protein expression is a prognostic factor for renal cell carcinoma patients.

The Piwi-like genes belong to the Argonaute gene family and are conserved in plants, animals and humans. In addition to their essential role in the germ line and as stem cell-associated genes, Piwi-like proteins play a role in different cancer types but have yet to be studied in renal cell carcinoma (RCC). We investigated tissue micro arrays (TMAs) with tumor samples of two independent cohorts of RCC patients (N = 265 and N = 345); we used immunohistochemistry to assess the protein expression of Piwi-like 1. Applying an immunoreactive score (IRS), we found Piwi-like 1 positivity (IRS > 0) in 28.3% and 14.8% of the tumors in cohorts 1 and 2, respectively. Piwi-like 1 positivity was correlated with Fuhrman grade, tumor stage and the presence of distant metastasis (P < 0.005). Moreover, in univariate and multivariate analyses (adjusted to Fuhrman grade and tumor stage), Piwi-like 1 positivity was associated with a shorter cancer-specific survival in the patients in the second cohort. In addition, Piwi-like 1 expression allowed to further distinguish the RCC patients with high Fuhrman grade, high tumor stage, distant metastasis or high pre-operative levels of C-reactive protein, as Piwi-like 1 positivity was associated with a shorter cancer-specific survival in both cohorts. Our data encourage further investigations to enlighten the role of Piwi-like 1 and its function as a marker of poor prognosis in RCC patients.

Expression of GP88 (Progranulin) Protein Is an Independent Prognostic Factor in Prostate Cancer Patients.

Prostate cancer, the second most common cancer, is still a major cause of morbidity and mortality among men worldwide. The expression of the survival and proliferation factor progranulin (GP88) has not yet been comprehensively studied in PCa tumors. The aim of this study was to characterize GP88 protein expression in PCa by immunohistochemistry and to correlate the findings to the clinico-pathological data and prognosis. Immunohistochemical staining for GP88 was performed by TMA with samples from 442 PCa patients using an immunoreactive score (IRS). Altogether, 233 cases (52.7%) with negative GP88 staining (IRS < 2) and 209 cases (47.3%) with positive GP88 staining (IRS ≥ 2) were analyzed. A significant positive correlation was found for the GP88 IRS with the PSA value at prostatectomy and the cytoplasmic cytokeratin 20 IRS, whereas it was negatively associated with follow-up times. The association of GP88 staining with prognosis was further studied by survival analyses (Kaplan-Meier, univariate and multivariate Cox's regression analysis). Increased GP88 protein expression appeared as an independent prognostic factor for overall, disease-specific and relapse-free survival in all PCa patients. Interestingly, in the subgroup of younger PCa patients (≤65 years), GP88 positivity was associated with a 3.8-fold (p = 0.004), a 6.0-fold (p = 0.008) and a 3.7-fold (p = 0.003) increased risk for death, disease-specific death and occurrence of a relapse, respectively. In the PCa subgroup with negative CK20 staining, GP88 positivity was associated with a 1.8-fold (p = 0.018) and a 2.8-fold increased risk for death and disease-specific death (p = 0.028). Altogether, GP88 protein positivity appears to be an independent prognostic factor for PCa patients.

Piwi-like 1 and -2 protein expression levels are prognostic factors for muscle invasive urothelial bladder cancer patients.

Piwi-like proteins are essential for stem-cell maintenance and self-renewal in multicellular organisms. We analyzed the expression of Piwi-like 1 and Piwi-like 2 by immunohistochemistry (IHC) in 95 muscle invasive bladder cancer (MIBC) samples using tissue microarray. Application of an immunoreactive score (IRS) revealed 37 and 45 patients who were Piwi-like 1 and -2 positive (IRS > 2). IHC results were correlated with clinico-pathological and survival data. The expression of both proteins was positively correlated with each other, lymph node metastasis and expression of CK20 and GATA 3. A negative correlation for both proteins was detected for disease-specific survival (DSS), recurrence, Ki67/MIB1 proliferation index, and CK5 expression. Detection of Piwi-like 1 protein positivity was associated with poor DSS (P = 0.019; log rank test, Kaplan-Meier analysis), and in multivariate Cox's analysis (adjusted to tumor stage and tumor grade), it was an independent prognostic factor for DSS (RR = 2.16; P = 0.011). Piwi-like 2 positivity was associated with DSS (P = 0.008) and recurrence-free survival (RFS; P = 0.040), and in multivariate Cox's analysis, Piwi-like 2 positivity was an independent prognostic factor for DSS (RR = 2.46; P = 0.004) and RFS (RR = 3.0; P = 0.003). Most interestingly, in the basal type patient subgroup (CK5+/GATA3-), Piwi-like 2 positivity was associated with poorer DSS, OS and RFS (P < 0.001, P = 0.004 and P = 0.05; log rank test). In multivariate analysis, Piwi-like 2 positivity was an independent prognostic factor for DSS (RR = 12.70; P = 0.001), OS (RR = 6.62; = 0.008) and RFS (RR=13.0; P = 0.040). In summary, Piwi-like 1 and -2 positivity are associated with clinico-pathological factors and survival. Both Piwi-like proteins are suggested as biomarkers for MIBC patients.

Differential prognostic value of MYC immunohistochemistry in subtypes of papillary renal cell carcinoma.

The histomorphological subtyping of papillary renal cell carcinomas (pRCCs) has improved the predictions of patients' long-term survival. Based on our previous results, we hypothesized that the MYC proto-oncogene would show differential expression in pRCC subtypes. Using a multi-institutional cohort of 204 pRCC patients we assessed the additional value of the immunohistochemical markers MYC, MINA53, and Ki67 in predicting patient's long-term survival. The clinical endpoints were overall survival (OS) and cancer-specific survival (CSS). Nomograms were constructed to predict each patient's risk of death (OS). The incorporation of the MYC staining patterns allowed the stratification of pRCC type 1 patients into better and worse prognostic groups. None of the patients with pRCC type 1 tumors and favorable MYC staining patterns died from tumor-related causes. This prognostic value was independent of the patient's age at surgery, the pathological tumor stage and presence of lymph node invasion. we could show that the immunohistochemical assessment of MYC and the histomorphological subtyping of pRCC stratifies pRCC type 1 tumors with regard to OS and CSS. The determination of the histomorphologic pRCC subtype in combination with the MYC immunohistochemical staining patterns allows a more accurate prediction of patients' individual risk of death.

Inactivation of the LKB1-AMPK signaling pathway does not contribute to salivary gland tumor development - a short report.

PURPOSE: Activation of AMPK by the tumor suppressor LKB1 represents an essential gatekeeping step for cells under energetic stress to prevent their growth and proliferation by inhibiting mTOR activation, until the energy supply normalizes. The LKB1/AMPK pathway is frequently downregulated in various types of cancer, thereby uncoupling tumor cell growth and proliferation from energy supply. As yet, little information is available on the role of the LKB1/AMPK pathway in tumors derived from salivary gland tissues. METHODS: We performed LKB1 protein expression and AMPK and mTOR activation analyses in several salivary gland tumor types and their respective healthy control tissues using immunohistochemistry. RESULTS: No significant downregulation of LKB1 expression or decreased activation of AMPK or mTOR were observed in any of the salivary gland tumors tested. In contrast, we found that the salivary gland tumors exhibited an increased rather than a decreased AMPK activation. Although the PI3K/Akt pathway was found to be activated in most of the analyzed tumor samples, the unchanged robust activity of LKB1/AMPK likely prevents (over)activation of mTOR. CONCLUSION: In contrast to many other types of cancer, inactivation or downregulation of the LKB1/AMPK pathway does not substantially contribute to the pathogenesis of salivary gland tumors.

Piwil 2 expression is correlated with disease-specific and progression-free survival of chemotherapy-treated bladder cancer patients.

Piwi-like 2 (Piwil 2) belongs to the family of Argonaute genes/proteins. The expression of Piwil 2 is associated with stem cells. A role in tumorigenesis and/or tumor progression is proposed for different cancers but not yet for bladder cancer (BCa). We investigated the Piwil 2 expression by immunohistochemistry in a cohort of 202 BCa patients treated by cystectomy and adjuvant chemotherapy. The association between Piwil 2 expression and disease-specific (DSS) or progression-free survival (PFS) was calculated using Kaplan Meier analyses and univariate/multivariate Cox's regression hazard models.In a multivariate Cox's regression, Piwil 2 expression, either in the cytoplasm or the nucleus, was significantly associated with DSS and PFS. A weak cytoplasmic staining pattern was associated with poor DSS and tumor progression (RR=2.7; P=0.004 and RR=2.4; P=0.027). Likewise,, absent nuclear Piwil 2 immunoreactivity was associated with poor DSS and tumor progression (RR=2.3; P=0.023 and RR=2.2; P=0.022). BCa patients whose tumors exhibited a combination of weak cytoplasmic and absent nuclear immunoreactivity had a 6-fold increased risk of tumor-related death (P=0.005) compared to patients with strong expression. Considering only patients with high grade G3 tumors, a 7.8-fold risk of tumor-associated death and a 3.6-fold risk of tumor progression were detected independently of the histologic tumor subtype or the chemotherapy regimen. In summary, a combination of weak cytoplasmic and absent nuclear expression of Piwil 2 is significantly associated with an increased risk of DSS and tumor progression. This implicates that Piwil 2 could be a valuable prognostic marker for high-risk BCa patients.

Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization.

Endosperm development in maize (Zea mays L.) and related cereals comprises a cell proliferation stage followed by a period of rapid growth coupled to endoreduplication. Regulation of the cell cycle in developing endosperm is poorly understood. We have characterized various subunits of cyclin-dependent kinase (CDK) complexes, master cell cycle regulators in all eukaryotes. A-, B-, and D-type cyclins as well as A- and B-type cyclin-dependent kinases were characterized with respect to their RNA and protein expression profiles. Two main patterns were identified: one showing expression throughout endosperm development, and another characterized by a sharp down-regulation with the onset of endoreduplication. Cyclin CYCB1;3 and CYCD2;1 proteins were distributed in the cytoplasm and nucleus of cells throughout the endosperm, while cyclin CYCD5 protein was localized in the cytoplasm of peripheral cells. CDKB1;1 expression was strongly associated with cell proliferation. Expression and cyclin-binding patterns suggested that CDKA;1 and CDKA;3 are at least partially redundant. The kinase activity associated with the cyclin CYCA1 was highest during the mitotic stage of development, while that associated with CYCB1;3, CYCD2;1 and CYCD5 peaked at the mitosis-to-endoreduplication transition. A-, B- and D-type cyclins were more resistant to proteasome-dependent degradation in endoreduplicating than in mitotic endosperm extracts. These results indicated that endosperm development is characterized by differential expression and activity of specific cyclins and CDKs, and suggested that endoreduplication is associated with reduced cyclin proteolysis via the ubiquitin-proteasome pathway.

Identification and characterization of lysine-rich proteins and starch biosynthesis genes in the opaque2 mutant by transcriptional and proteomic analysis.

BACKGROUND: The opaque2 mutant is valuable for producing maize varieties with enhanced nutritional value. However, the exact mechanisms by which it improves protein quality and creates a soft endosperm texture are unclear. Given the importance of improving nutritional quality in grain crops, a better understanding of the physiological basis for these traits is necessary. RESULTS: In this study, we combined transcript profiling and proteomic analysis to better understand which genes and proteins are altered by opaque2 in the W64A inbred line. These analyses showed that the accumulation of some lysine-rich proteins, such as sorbitol dehydrogenase and glyceraldehyde3-phosphate dehydrogenase, was increased in mature kernels and may contribute substantially to the lysine content of opaque2 endosperm. Some defense proteins such as beta-glucosidase aggregating factor were strongly down regulated and may be regulated directly by opaque2. The mutant also had altered expression of a number of starch biosynthesis genes and this was associated with a more highly crystalline starch. CONCLUSIONS: The results of these studies revealed specific target genes that can be investigated to further improve nutritional quality and agronomic performance of high lysine maize lines, particularly those based on the presence of the opaque2 mutation. Alteration of amylopectin branching patterns in opaque2 starch could contribute to generation of the soft, starchy endosperm.

Clinical significance of histone deacetylases 1, 2, 3, and 7: HDAC2 is an independent predictor of survival in HCC.

Histone deacetylases (HDAC) are responsible for the transcriptional control of genes through chromatin remodeling and control tumor suppressor genes. In several tumors, their expression has been linked to clinicopathological factors and patient survival. This study investigates HDACs 1, 2, 3, and 7 expressions in hepatocellular carcinoma (HCC) and their correlation with clinical data and patient survival. Tissue microarrays of 170 surgically resected primary HCCs and adjacent uninvolved tissue were evaluated immunohistochemically for the expression of HDACs 1, 2, 3, 7, and Ki-67 and were analyzed with respect to clinicopathological data and patient survival. HDACs 1, 2, 3, and Ki-67 were expressed significantly higher in cancer cells compared to normal tissue (HDAC1: p = 0.034, HDACs 2 and 3 and Ki-67: p < 0.001), while HDAC7 expression did not differ between HCC and non-cancerous liver tissue. In tumor tissue HDACs 1-3 expression levels showed high concordance with each other, Ki-67 and tumor grade (p < 0.001). High HDAC2 expression was associated with poor survival in low-grade and early-stage tumors (p < 0.05). The expression of the HDACs 1, 2, and 3 (but not HDAC7) isoenzymes correlates with clinicopathological factors, and HDAC2 expression has an impact on patient survival.

Expression and localization of the uptake transporters OATP2B1, OATP3A1 and OATP5A1 in non-malignant and malignant breast tissue.

Organic anion transporting polypeptides (OATPs, gene family SLCO/SLC21) mediate the uptake of multiple endogenous substances such as estrogens and estrogen metabolites and of several widely prescribed drugs (e.g. statins, antibiotics and anticancer agents) into cells. Since several anticancer agents have been identified as substrates for OATPs, these transporters may also have an impact on cancer treatment. Expression of OATPs has been identified in colon, pancreatic and gastric carcinomas but to date little is known about the expression and localization of OATP family members in non-malignant breast tissue and breast cancer. We therefore analyzed the mRNA expression of all human OATP family members and further evaluated the mRNA amount of the highly-expressed OATPs OATP2B1, OATP3A1 and OATP5A1 in 10 paired samples of normal breast tissue and breast cancer. Furthermore, the tissue-specific localization of these OATPs was investigated. The results demonstrated that the mRNA expression of OATPs in normal and malignant breast tissue shows a high interindividual variability and that no significant differences in the mRNA amount between normal and malignant tissue could be detected. Furthermore, we localized OATP3A1 and OATP5A1 to the plasma membrane of epithelial cells of the lactiferous ducts in normal breast tissue. In breast cancer, both OATPs are highly expressed in the plasma membrane and in the cytoplasm. Since estrogen and estrogen metabolites as well as anticancer agents are substrates for OATPs these results indicate the possibility of OATP-mediated uptake of hormones during breast cancer development and an impact of certain OATPs on chemotherapeutic cancer treatment.

Delivery of prolamins to the protein storage vacuole in maize aleurone cells.

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.

Characterization of opaque2 modifier QTLs and candidate genes in recombinant inbred lines derived from the K0326Y quality protein maize inbred.

Quality protein maize (QPM) is a high lysine-containing corn that is based on genetic modification of the opaque2 (o2) mutant. In QPM, modifier genes convert the starchy endosperm of o2 to the vitreous phenotype of wild type maize. There are multiple, unlinked o2 modifier loci (Opm) in QPM and their nature and mode of action are unknown. We previously identified seven Opm QTLs and characterized 16 genes that are differentially up-regulated at a significant level in K0326Y QPM, compared to the starchy endosperm mutant W64Ao2. In order to further characterize these Opm QTLs and the genes up-regulated in K0326Y QPM, we created a population of 314 recombinant inbred lines (RILs) from a cross between K0326Y QPM and W64Ao2. The RILs were characterized for three traits associated with endosperm texture: vitreousness, density and hardness. Genetic linkage analysis of the RIL population confirmed three of the previously identified QTLs associated with o2 endosperm modification in K0326Y QPM. Many of the genes up-regulated in K0326Y QPM showed substantially higher levels of expression in vitreous compared with opaque RILs. These included genes associated with the upstream regulation of the ethylene response pathway, and a gene encoding a regulatory subunit of pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase, an adaptive enzyme of the glycolytic pathway.