Enhanced Clinical Utility of Molecular Budding Signature as a Recurrence Risk Determinant in Stage II and III Colon Cancer Patients.

BACKGROUND: A molecular budding signature (MBS), which consists of seven tumor budding-related genes, was recently presented as a prominent prognostic indicator in colon cancer (CC) using microarray data acquired from frozen specimens. This study aimed to confirm the predictive power of MBS for recurrence risk based on formalin-fixed, paraffin-embedded (FFPE) materials. METHODS: This research utilized the same microarray data from a prior multicenter study using FFPE whole tissue sections, which retrospectively reviewed 232 stage II CC patients without adjuvant chemotherapy and 302 stage III CC patients with adjuvant chemotherapy. All patients underwent upfront curative surgery without neoadjuvant therapy between 2009 and 2012. An MBS score was calculated using the mean of log2 [each signal] of seven genes (MSLN, SLC4A11, WNT11, SCEL, RUNX2, MGAT3, and FOXC1) as described before. RESULTS: The MBS-low group exhibited a better relapse-free survival (RFS) than the MBS-high group in stage II (P = 0.0077) and in stage III CC patients (P = 0.0003). Multivariate analyses revealed that the MBS score was an independent prognostic factor in both stage II (P = 0.0257) and stage III patients (P = 0.0022). Especially among T4, N2, or both (high-risk) stage III patients, the MBS-low group demonstrated markedly better RFS compared with the MBS-high group (P = 0.0013). CONCLUSIONS: This study confirmed the predictive power of the MBS for recurrence risk by employing FFPE materials in stage II/III CC patients.

Establishing a whole blood CD4+ T cell immunity measurement to predict response to anti-PD-1.

BACKGROUND: Biomarkers that can accurately predict the efficacy of immune checkpoint inhibitors (ICIs) against programmed death 1 (PD-1) ligand in cancer immunotherapy are urgently needed. We have previously reported a novel formula that predicts the response to treatment with second-line nivolumab with high sensitivity and specificity in patients with non-small cell lung cancer (NSCLC) previously treated with chemotherapy. The formula was based on the percentages of CD62LlowCD4+ T cells (effector T cells; %Teff) and CD4+CD25+FOXP3+ T cells (regulatory T cells; %Treg) in the peripheral blood before treatment estimated using the peripheral blood mononuclear cell (PBMC) method. Here, we investigated the applicability of the formula (K-index) to predict the response to treatment with another ICI to expand its clinical applicability. Furthermore, we developed a simpler assay method based on whole blood (WB) samples to overcome the limitations of the PBMC method, such as technical difficulties, in obtaining the K-index. METHODS: The K-index was evaluated using the PBMC method in 59 patients with NSCLC who received first-line pembrolizumab treatment. We also assessed the K-index using the WB method and estimated the correlation between the measurements obtained using both methods in 76 patients with lung cancer. RESULTS: This formula consistently predicted the response to first-line pembrolizumab therapy in patients with NSCLC. The WB method correlated well with the PBMC method to obtain %Teff, %Treg, and the formula value. The WB method showed high repeatability (coefficient of variation, < 10%). The data obtained using WB samples collected in tubes containing either heparin or EDTA-2K and stored at room temperature (18-24 °C) for one day after blood sampling did not differ. Additionally, the performance of the WB method was consistent in different flow cytometry instruments. CONCLUSIONS: The K-index successfully predicted the response to first-line therapy with pembrolizumab, as reported earlier for the second-line therapy with nivolumab in patients with NSCLC. The WB method established in this study can replace the cumbersome PBMC method in obtaining the K-index. Overall, this study suggests that the K-index can predict the response to anti-PD-1 therapy in various cancers, including NSCLC.

A 95-gene signature stratifies recurrence risk of invasive disease in ER-positive, HER2-negative, node-negative breast cancer with intermediate 21-gene signature recurrence scores.

PURPOSE: A subset of patients with intermediate 21-gene signature assay recurrence score may benefit from adjuvant chemoendocrine therapy, but a predictive strategy is needed to identify such patients. The 95-gene signature assay was tested to stratify patients with intermediate RS into high (95GC-H) and low (95GC-L) groups that were associated with invasive recurrence risk. METHODS: Patients with ER-positive, HER2-negative, node-negative breast cancer and RS 11-25 who underwent definitive surgery and adjuvant endocrine therapy without any cytotoxic agents were included. RNA was extracted from archived formalin-fixed, paraffin-embedded samples, and 95-gene signature was calculated. RESULTS: 206 patients had RS of 11-25 (95GC-L, N = 163; 95GC-H, N = 43). In Cox proportional hazards model, 95GC-H was significantly associated with shorter time to recurrence than was 95GC-L (HR 5.94; 95%CI 1.81-19.53; P = 0.005). The correlation between 95-gene signature and 21-gene signature assay scores was not strong (correlation coefficient r = 0.27), which might suggest that 95-gene signature reflects biological characteristics differing from what 21-gene signature shows. CONCLUSIONS: The 95-gene signature stratifies patients with ER-positive, HER2-negative, node-negative invasive breast cancer and intermediate RS of 11-25 into high and low groups that are associated with recurrence risk of invasive disease. Further retrospective analysis in the prospectively accrued TAILORx population is warranted to confirm that 95-gene signature can identify patients who would benefit from adjuvant chemoendocrine therapy.

Evaluation of a 55-gene classifier as a prognostic biomarker for adjuvant chemotherapy in stage III colon cancer patients.

BACKGROUND: Adjuvant chemotherapy reduces the risk of recurrence of stage III colon cancer (CC). However, more effective prognostic and predictive biomarkers are needed for better treatment stratification of affected patients. Here, we constructed a 55-gene classifier (55GC) and investigated its utility for classifying patients with stage III CC. METHODS: We retrospectively identified patients aged 20-79 years, with stage III CC, who received adjuvant chemotherapy with or without oxaliplatin, between the years 2009 and 2012. RESULTS: Among 938 eligible patients, 203 and 201 patients who received adjuvant chemotherapy with and without oxaliplatin, respectively, were selected by propensity score matching. Of these, 95 patients from each group were analyzed, and their 5-year relapse-free survival (RFS) rates with and without oxaliplatin were 73.7 and 77.1%, respectively. The hazard ratios for 5-year RFS following adjuvant chemotherapy (fluoropyrimidine), with and without oxaliplatin, were 1.241 (95% CI, 0.465-3.308; P = 0.67) and 0.791 (95% CI, 0.329-1.901; P = 0.60), respectively. Stratification using the 55GC revealed that 52 (27.3%), 78 (41.1%), and 60 (31.6%) patients had microsatellite instability (MSI)-like, chromosomal instability (CIN)-like, and stromal subtypes, respectively. The 5-year RFS rates were 84.3 and 72.0% in patients treated with and without oxaliplatin, respectively, for the MSI-like subtype (HR, 0.495; 95% CI, 0.145-1.692; P = 0.25). No differences in RFS rates were noted in the CIN-like or stromal subtypes. Stratification by cancer sidedness for each subtype showed improved RFS only in patients with left-sided primary cancer treated with oxaliplatin for the MSI-like subtype (P = 0.007). The 5-year RFS rates of the MSI-like subtype in left-sided cancer patients were 100 and 53.9% with and without oxaliplatin, respectively. CONCLUSIONS: Subclassification using 55GC and tumor sidedness revealed increased RFS in patients within the MSI-like subtype with stage III left-sided CC treated with fluoropyrimidine and oxaliplatin compared to those treated without oxaliplatin. However, the predictive power of 55GC subtyping alone did not reach statistical significance in this cohort, warranting larger prospective studies. TRIAL REGISTRATION: The study protocol was registered in the University Hospital Medical Education Network (UMIN) clinical trial registry (UMIN study ID: 000023879 ).

A Validation Study for Recurrence Risk Stratification of Stage II Colon Cancer Using the 55-Gene Classifier.

INTRODUCTION: DNA microarrays, such as the consensus molecular subtype (CMS) classification using >600 genes, are used to predict cancer patient prognosis. We recently constructed a simple 55-gene classifier (55GC) system to risk stratify colon cancer (CC). OBJECTIVE: Here, we validate the 55GC specifically for stage II CC and compare it with CMS categories. METHODS: Tissue sections from 232 stage II CC patients who underwent curative surgery without adjuvant chemotherapy between 2009 and 2012 were subjected to DNA microarray analysis. RESULTS: Based on the 55GC, patients were classified into microsatellite instability-like (27%), chromosomal instability-like (41%), and stromal (32%) subtypes with 5-year relapse-free survival (RFS) rates of 88.5, 83.3, and 71.2%, respectively (stromal vs. others: p = 0.0049). Multivariate analysis by Cox's proportional hazard model revealed that the stromal subtype, pT4, and the number of lymph nodes examined (<12) were independent poor prognostic factors. The overall concordance rate between 55GC and CMS was 72%, and 5-year RFS rates of patients with CMS1, CMS2, CMS3, and CMS4 cancers were 100, 85.5, 92.3, and 73.0%, respectively (p = 0.0113). CONCLUSIONS: We conclude that the 55GC is a useful and reproducible grading system for stage II CC recurrence risk stratification.

Cyclin-Dependent Kinase Activity Correlates with the Prognosis of Patients Who Have Gastrointestinal Stromal Tumors.

BACKGROUND: The estimation of recurrence risk remains a critical issue in relation to gastrointestinal stromal tumors (GISTs) treated with adjuvant therapy. The accuracy of the commonly used risk stratifications is not always adequate. METHODS: For this study, data were prospectively collected from 68 patients with GISTs who underwent R0 surgery between 2004 and 2009. The results from this analysis cohort were evaluated using the data obtained from an additional 40 patients in the validation cohort. Cyclin-dependent kinase 1 (CDK1)- and CDK2-specific activities were measured using a non-RI kinase assay system. RESULTS: The specific activities of CDK1 and CDK2, but not their expression, significantly correlated with recurrence. The specific activities of both CDK1 and CDK2 were independently correlated with mitosis and significantly correlated with recurrence-free survival (RFS). In the multivariate analysis, CDK2-specific activity (P = 0.0006), tumor size (P = 0.0347), and KIT deletion mutations (P = 0.0006) were significantly correlated with RFS in the analysis cohort. In the validation cohort, CDK2-specific activity (P = 0.0368) was identified as an independent prognostic factor for tumor recurrences with tumor location (P = 0.0442). CONCLUSION: The results suggest that the specific activities of CDK1 and CDK2 may reflect the proliferative activity of GISTs and that CDK2-specific activity is a good prognostic factor predicting recurrence after macroscopic complete resection of GISTs.

Cyclin-dependent kinase-specific activity predicts the prognosis of stage I and stage II non-small cell lung cancer.

BACKGROUND: Lung cancer is one of the leading causes of cancer death worldwide. Even with complete resection, the prognosis of early-stage non-small cell lung cancer is poor due to local and distant recurrence, and it remains unclear which biomarkers are clinically useful for predicting recurrence or for determining the efficacy of chemotherapy. Recently, several lines of evidence have indicated that the enzymatic activity of cyclin-dependent kinases could be a clinically relevant prognostic marker for some cancers. We investigated whether the specific activity of cyclin-dependent kinases 1 and 2 could predict recurrence or death in early non-small cell lung cancer patients. METHODS: Patients with newly diagnosed, pathologically confirmed non-small cell lung cancer were entered into this blinded cohort study. The activity of cyclin-dependent kinases was determined in 171 samples by the C2P® assay, and the results were subjected to statistical analysis with recurrence or death as a clinical outcome. RESULTS: The Cox proportional hazards model revealed that the activity of cyclin-dependent kinase 1, but not 2, was a predictor of recurrence, independent of sex, age, and stage. By contrast, cyclin-dependent kinase 2 activity was a predictor of death, independent of sex and stage. CONCLUSION: This study suggested the possible clinical use of cyclin-dependent kinase 1 as a predictor of recurrence and cyclin-dependent kinase 2 as a predictor of overall survival in early-stage non-small cell lung cancer. Thus, a combination of activity of cyclin-dependent kinases 1 and 2 is useful in decision-making regarding treatment strategies for non-small cell lung cancer after surgery.

The prognostic value of apoptotic and proliferative markers in breast cancer.

Increasing ability of early breast cancer (BC) diagnosis leading to more early stage detection, better survival, and low relapse marks one of the milestones achieved over the decades. Foregoing poses a challenge for clinicians regarding optimal treatment, in which over- and under-treatment should be avoided. Classical prognostic and predictive factors fall short for individualized adjuvant therapy selection in this patient group. The key to better characterization may be found in the biology underlying individual tumors. We hypothesized that markers related to cellular proliferation and apoptosis and the balance between these two processes in tumor development will be predictive for clinical outcome. Our study population (N = 822) consisted of all early stage BC patients primarily treated with surgery in our center between 1985 and 1996. Sections of available tumor tissue (87 %, 714/822) were immunohistochemically stained for expression of p53, active-caspase-3, and Ki67. In 43 % (304/714) and 18 % (126/714) of this cohort, respectively, a biochemical C2P(®) risk prediction and caspase-3 assay were performed. Expression data of the mentioned markers, single, or combined, were analyzed. Results showed that both the single and combined markers, whether of apoptotic or proliferative origin had associations with clinical outcome. An additive effect was seen for the hazard ratios when data on p53, active caspase-3, and Ki67 status were combined. The assembled prognostic apoptotic-proliferative subtype showed significant association for both the overall survival (p = 0.024) and relapse-free period (p = 0.001) in the multivariate analyses of grade I breast tumors. Combined markers of tumor cell apoptosis and proliferation represent tumor aggressiveness. The apoptotic-proliferative subtypes that we present in this study represent a clinical prognostic profile with solid underlying biological rationale and pose a promising method for accurate identification of grade I BC patients in need of an aggressive therapeutic approach, thus contributing to precision medicine in BC disease.

Correlates of neutralizing/SARS-CoV-2-S1-binding antibody response with adverse effects and immune kinetics in BNT162b2-vaccinated individuals.

While mRNA vaccines against SARS-CoV-2 are exceedingly effective in preventing symptomatic infection, their immune response features remain to be clarified. In the present prospective study, 225 healthy individuals in Japan, who received two BNT162b2 doses, were enrolled. Correlates of BNT162b2-elicited SARS-CoV-2-neutralizing activity (50% neutralization titer: NT50; assessed using infectious virions) with various determinants were examined and the potency of sera against variants of concerns was determined. Significant rise in NT50s was seen in sera on day 28 post-1st dose. A moderate inverse correlation was seen between NT50s and ages, but no correlation seen between NT50s and adverse effects. NT50s and SARS-CoV-2-S1-binding-IgG levels on day 28 post-1st dose and pain scores following the 2nd dose were greater in women than in men. The average half-life of NT50s was ~ 68 days, and 23.6% (49 out of 208 individuals) failed to show detectable neutralizing activity on day 150. While sera from elite-responders (NT50s > 1,500: the top 4% among the participants) potently to moderately blocked all variants of concerns examined, some sera with low NT50s failed to block the B.1.351-beta strain. Since BNT162b2-elicited immunity against SARS-CoV-2 is short, an additional vaccine or other protective measures are needed.

Correlates of Neutralizing/SARS-CoV-2-S1-binding Antibody Response with Adverse Effects and Immune Kinetics in BNT162b2-Vaccinated Individuals.

BACKGROUND: While mRNA vaccines against SARS-CoV-2 have been exceedingly effective in preventing symptomatic viral infection, the features of immune response remain to be clarified. METHODS: In the present prospective observational study, 225 healthy individuals in Kumamoto General Hospital, Japan, who received two BNT162b2 doses in February 2021, were enrolled. Correlates of BNT162b2-elicited SARS-CoV-2-neutralizing activity (50% neutralization titer: NT 50 ; assessed using infectious virions and live target cells) with SARS-CoV-2-S1-binding-IgG and -IgM levels, adverse effects (AEs), ages, and genders were examined. The average half-life of neutralizing activity and the average time length for the loss of detectable neutralizing activity were determined and the potency of serums against variants of concerns was also determined. FINDINGS: Significant rise in NT 50 s was seen in serums on day 28 post-1st dose. A moderate inverse correlation was seen between NT 50 s and ages, but no correlation was seen between NT 50 s and AEs. NT 50 s and IgG levels on day 28 post-1st dose and pain scores following the 2nd shot were greater in women than in men. The average half-life of neutralizing activity in the vaccinees was approximately 67.8 days and the average time length for their serums to lose the detectable neutralizing activity was 198.3 days. While serums from elite-responders (NT 50 s>1,500-fold: the top 4% among all participants' NT 50 s) potently to moderately blocked the infectivity of variants of concerns, some serums with moderate NT 50 s failed to block the infectivity of a beta strain. INTERPRETATION: BNT162b2-elicited immune response has no significant association with AEs. BNT162b2-efficacy is likely diminished to under detection limit by 6-7 months post-1st shot. High-level neutralizing antibody-containing serums potently to moderately block the infection of SARS-CoV-2 variants; however, a few moderate-level neutralizing antibody-containing serums failed to do so. If BNT162b2-elicited immunity memory is short, an additional vaccine or other protective measures would be needed. RESEARCH IN CONTEXT: Evidence before this study: While mRNA vaccines against SARS-CoV-2 have been exceedingly effective in preventing symptomatic viral infection, the salient features of immune response including the persistence of protection remain to be clarified. There is a report that anti-SARS-CoV-2 antibodies persist through 6 months after the second dose of mRNA-1273 vaccine (Doria-Rose et al. N Engl J Med . 2021;384:2259-2261); however, more definite immune kinetics following mRNA-vaccine-elicited protection have to be clarified. The mRNA-vaccine-elicited protection against SARS-CoV-2 variants are also to be determined. Added value of this study: In the present prospective study, 225 twice-BNT162b2-dose-receiving individuals in Japan were enrolled. No significant correlation was seen between 50% neutralizing titers (NT 50 s), determined by using infectious SARS-CoV-2 virions and live target cells, and adverse effects. Largely, NT 50 s and IgG levels were greater in women than in men. Following 28 days post-2 nd shot, significant reduction was seen in NT 50 s, IgG, and IgM levels. The average half-life of NT 50 s was ∼68 days and the average time-length for participants' serums to lose the detectable activity was ∼198 days. Although serums from elite-responders potently to moderately blocked the infectivity of variants of concerns, some serums with moderate NT 50 s failed to block the infectivity of a beta strain. Implications of all the available evidence: BNT162b2 efficacy is likely to be diminished to under detection limit by 6-7 months post-1 st shot on average. Individuals with moderate NT 50 s may fail to block beta variants. If BNT162b2-elicited immune memory is lost soon, additional vaccine(s) or other protective means would be needed.

Prediction of paclitaxel sensitivity by CDK1 and CDK2 activity in human breast cancer cells.

INTRODUCTION: Paclitaxel is used widely in the treatment of breast cancer. Not all tumors respond to this drug, however, and the characteristics that distinguish resistant tumors from sensitive tumors are not well defined. Activation of the spindle assembly checkpoint is required for paclitaxel-induced cell death. We hypothesized that cyclin-dependent kinase (CDK) 1 activity and CDK2 activity in cancer cells, which reflect the activation state of the spindle assembly checkpoint and the growth state, respectively, predict sensitivity to paclitaxel. METHODS: Cell viability assays and DNA and chromatin morphology analyses were performed in human breast cancer cell lines to evaluate sensitivity to paclitaxel and the cell cycle response to paclitaxel. We then examined the specific activities of CDK1 and CDK2 in these cell lines and in xenograft models of human breast cancer before and after paclitaxel treatment. Protein expression and kinase activity of CDKs and cyclins were analyzed using a newly developed assay system. RESULTS: In the cell lines, biological response to paclitaxel in vitro did not accurately predict sensitivity to paclitaxel in vivo. Among the breast cancer xenograft tumors, however, tumors with significantly increased CDK1 specific activity after paclitaxel treatment were sensitive to paclitaxel in vivo, whereas tumors without such an increase were resistant to paclitaxel in vivo. Baseline CDK2 specific activity was higher in tumors that were sensitive to paclitaxel than in tumors that were resistant to paclitaxel. CONCLUSIONS: The change in CDK1 specific activity of xenograft tumors after paclitaxel treatment and the CDK2 specific activity before paclitaxel treatment are both associated with the drug sensitivity in vivo. Analysis of cyclin-dependent kinase activity in the clinical setting could be a powerful approach for predicting paclitaxel sensitivity.

A new cancer diagnostic system based on a CDK profiling technology.

A series of molecular pathological investigations of the molecules that stimulate the cyclin dependent kinases (CDK1, 2, 4, and 6) have led to enormous accumulation of knowledge of the clinical significance of these molecules for cancer diagnosis. However, the molecules have yet to be applied to clinical cancer diagnosis, as there is no available technology for application of the knowledge in a clinical setting. We hypothesized that the direct measurement of CDK activities and expressions (CDK profiling) might produce clinically relevant values for the diagnosis. This study investigated the clinical relevance of CDK profiling in gastrointestinal carcinoma tissues by using originally developed expression and activity analysis methods. We have established novel methods and an apparatus for analyzing the expression and activities of the CDK molecules in lysate of tumor tissue in a clinical setting, and examined 30 surgically dissected gastrointestinal carcinomas and corresponding normal mucosal specimens. We demonstrate here that remarkably elevated CDK2 activity is evident in more than 70% of carcinoma tissues. Moreover, a G1-CDK activity profiling accurately mirrored the differences in proliferation between tumor and normal colonic tissues. Our results suggest that CDK profiling is a potent molecular-clinical approach to complement the conventional pathological diagnosis, and to further assist in the individualized medications.

CDK1 and CDK2 activity is a strong predictor of renal cell carcinoma recurrence.

BACKGROUND: In renal cell carcinoma (RCC), the prediction of metastasis via tumor prognostic markers remains a major problem. The objective of our study was to evaluate the efficacy of cyclin-dependent kinase (CDK)1 and CDK2 activity as a prognostic marker in human RCC. METHODS: Surgical specimens were obtained from 125 patients with RCC without metastasis. Protein expression and kinase activity of CDKs were analyzed using a newly developed assay system named C2P (Sysmex, Kobe, Japan). We then examined the specific activities (SAs) of CDK1 and CDK2 and calculated CDK2SA-CDK1SA ratio in RCC. Also, risk score (RS) was examined. RESULTS: A total of 125 cases were tested, though 34 cases were excluded because of low sample quality (25 cases) and assay failure (9 cases). In total, 91 cases were analyzed. They included 68 male and 23 female patients, ranging in age from 19 to 83 years. At a median follow-up of 36 months (1-109M), tumor with low CDK2SA-CDK1SA ratio showed significantly better 5-year recurrence-free survival than those with high CDK2SA-CDK1SA ratio (88.7% vs. 54.7%, P = 0.00141). Also, RS enabled the classification of RCCs into high-risk and low-risk groups, and patients with tumors classified as low RS showed better recurrence-free survival than patients with tumors with high RS (88.7% vs. 54.7%, P = 0.0141). CONCLUSION: CDK1SA of tumors and the CDK2SA are both associated with recurrence and prognosis. IMPACT: CDK-based risk demonstrated is strongly associated with clinical outcome. CDK-based risk should be an accurate system for predicting recurrence and survival for planning follow-up.

Novel functional assay for spindle-assembly checkpoint by cyclin-dependent kinase activity to predict taxane chemosensitivity in breast tumor patient.

Taxanes are among the drugs most commonly used for preoperative chemotherapy for breast cancer. Taxanes induce mitotic arrest and subsequent apoptosis. The spindle-assembly checkpoint (SAC) is known to be activated during mitosis, along with cyclin-dependent kinase-1 (CDK1), and is required for taxane-induced cell death. We hypothesized that CDK1 activity predicts response to taxane-containing chemotherapy. This study included breast cancer patients who received preoperative chemotherapy- taxane-containing treatment followed by anthracycline-based treatment-and then underwent surgery. Before starting taxane-containing chemotherapy, patients underwent fine-needle aspiration biopsy, and the biopsy samples were incubated in paclitaxel solution to measure CDK activity. Clinical were evaluated after taxane therapy, and pathological resposes were evaluated after completion of all preoperative chemotherapy. Thirty five patients were eligible for analysis of clinical response to taxane-containing therapy. Twenty-six patients had taxane-sensitive and 9 taxane-resistant tumors. Using a cut-off of CDK activity determined by the ROC analysis, patients were classified into SAC function and dysfunction groups. Univariate logistic regression analysis with clinicopathologic parameters showed that only CDK-based SAC functionality was significantly correlated with clinical response (P =0.017). No significant correlation was observed between SAC functionality and pathologic response. CDK-based SAC functionality significantly predicted clinical response (P =.0072, overall agreement = 71.4%), and this is a unique mechanism-based marker for predicting taxane chemosensitivity. Further, large prospective study is needed to determine CDK-based SAC functionality could be developed as a predictive biomarker.

Cation diffusion facilitator Cis4 is implicated in Golgi membrane trafficking via regulating zinc homeostasis in fission yeast.

We screened for mutations that confer sensitivities to the calcineurin inhibitor FK506 and to a high concentration of MgCl(2) and isolated the cis4-1 mutant, an allele of the gene encoding a cation diffusion facilitator (CDF) protein that is structurally related to zinc transporters. Consistently, the addition of extracellular Zn(2+) suppressed the phenotypes of the cis4 mutant cells. The cis4 mutants and the mutant cells of another CDF-encoding gene SPBC16E9.14c (we named zrg17(+)) shared common and nonadditive zinc-suppressible phenotypes, and Cis4 and Zrg17 physically interacted. Cis4 localized at the cis-Golgi, suggesting that Cis4 is responsible for Zn(2+) uptake to the cis-Golgi. The cis4 mutant cells showed phenotypes such as weak cell wall and decreased acid phosphatase secretion that are thought to be resulting from impaired membrane trafficking. In addition, the cis4 deletion cells showed synthetic growth defects with all the four membrane-trafficking mutants tested, namely ypt3-i5, ryh1-i6, gdi1-i11, and apm1-1. Interestingly, the addition of extracellular Zn(2+) significantly suppressed the phenotypes of the ypt3-i5 and apm1-1 mutant cells. These results suggest that Cis4 forms a heteromeric functional complex with Zrg17 and that Cis4 is implicated in Golgi membrane trafficking through the regulation of zinc homeostasis in fission yeast.

Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist.

Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100< pmol/mouse) than when administered i.t. (ED(50): 1.34-4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.