Search

Background. Neoadjuvant chemotherapy (NACT) is increasingly administered for high-risk breast cancer patients. Yet, oncologists lack an effective method for assessing response to treatment beyond clinicpathological features. Organ tissue slices freshly obtained from the tumor and incubated in appropriate media, recapitulate the tumor’s heterogeneity and may provide a superior ex-vivo model for predicting response/resistance to treatment. While these models were tested in metastatic tumors, no studies up to date have been reported in early breast cancer or for NACT. These tests have short turnaround time (7-9 days), critical for the neoadjuvant setting. Here we aimed to prove the feasibility of performing cResponse assay, an ex vivo organ culture (EVOC), on freshly derived pre-treatment core needle biopsies and to evaluate it as a tool for predicting response to NACT in breast cancer. Methods. Pre-treatment tumor biopsies were obtained from breast cancer patients about to undergo NACT at the time of routine tumor clip marking. The fresh samples were immediately placed in cold medium, sliced into 250um sections and cultured in multiwell plates. The samples were treated with the clinically administered therapies or a vehicle control. The various therapies included Doxorubicin (A), Paclitaxel (T), Cytoxan (C) and in TN cases, Carboplatin (Cr), as single agents or in combination in clinically administered ratios. After 4 days of treatment, tumor slices were fixed and stained to allow morphological analysis. A trained pathologist examined the stained slides and scored the viable cancer cells. A response-score was generated and compared to the pathological result (pCR status and RCB class) of the patient at surgery.Results. Tumor samples from 15 breast cancer patients were examined. The cohort median age was 42 (32—82), LN involvement 9/15, subtypes status: 9 HR+, 2 TNBC, 3 HR+HER2+ and 1 HR-HER2+. Nine samples were either excluded due to insufficient viable cancer cells or necrosis and one sample failed technically. Five samples (2 TNBC, 2 HR+ and 1 HR+HER2+) were adequate for analysis and obtained cResponse scores. The HR+HER2+ case received a different protocol than actually tested, thus was not compared. For the four successful cases, the cResponse score was highly concordant with response to therapy (Table I). For the two TNBC samples, a maximal cResponse score (100) was demonstrated for AC-T or AC-TC combination and both patients achieved complete response (pCR, RCB-0) at surgery. Notably, in one patient, the assay predicted maximal response for the TC combination, suggesting that this protocol could be sufficient. For the HR+ patients, one patient demonstrated a strong cResponse to AC-T (85) and achieved near complete response (RCB-I), while the second patient demonstrated a moderate cResponse score (70) and achieved partial pathological response (RCB-II). Conclusions. We provide initial evidence for the feasibility and validity of an EVOC platform to predict response to NACT in breast cancer. Further optimizations are needed to increase the assay’s success rates. We expect the results to set the ground for a clinical trial, examining the utility of the cResponse test as predictive biomarker in determining NACT. This technology may provide a tool for the oncologist to select the most efficient therapies, maximizing pCR rates and minimizing toxicity from ineffective drugs, eventually improving prognosis of breast cancer patients. Table IPatientSubtypeBest cResponse scoreBest treatmentsPathological ResponseRCB classNAT-2HR+70AC-T/AC/CPartialRCB-IINAT-3TN100AC-TCr/TCrpCRRCB-0NAT-12TN100A/AC-TpCRRCB-0NAT-16HR+85AC-TPartialRCB-I Citation Format: Einav Nili Gal-Yam, Miri Sklair-Levi, Yaeli Vachnish, Nora Balint-Lahat, Dana Morzaev-Sulzbach, Michal Bakalenik-Gavry, Renata Fearmann, Osnat Halshtok, Anat Shalmon, Michael Gotlieb, Yael Yagil, Keren Levanon, Rinat Bernstein-Molho, Amit Itay, Tali Shapira-Rotenberg, Opher Globus, Iris Barshack, Seth Salpeter, Vered Bar, Sara Aharon, Lubov Turovsky, Adi Zundelevich, Giuseppe Mallel, Hamutal Shahar, Hagit Shapira, Maya Dadiani. Evaluating an ex vivo organ culture system for predicting response to neoadjuvant chemotherapy in breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-25.

Profiling tumor bacteria Bacteria are well-known residents in human tumors, but whether their presence is advantageous to the tumors or to the bacteria themselves has been unclear. As an initial step toward addressing this question, Nejman et al. produced an exhaustive catalog of the bacteria present in more than 1500 human tumors representing seven different tumor types (see the Perspective by Atreya and Turnbaugh). They found that the bacteria within tumors were localized within both cancer cells and immune cells and that the bacterial composition varied according to tumor type. Certain biologically plausible associations were identified. For example, breast cancer subtypes characterized by increased oxidative stress were enriched in bacteria that produce mycothiol, which can detoxify reactive oxygen species. Science , this issue p. 973 ; see also p. 938

Precision cancer therapy has the potential to revolutionize treatment outcome. While genomic analysis has become central to cancer personalized medicine, recent studies have not shown that it drastically improves patient survival as compared to standard drug selection. Additionally, genomic mutations may suggest several treatment protocols without elucidating which approach will yield the best clinical response. Moreover, for many drugs no genetic predictive biomarkers are available. To advance cancer precision diagnostics, we have developed cResponse, a functional drug sensitivity platform to determine individualized patient treatment regimens. Fresh patient cancer samples are taken by biopsy or resection and sectioned into 300 uM slices which when cultured in the cResponse platform demonstrate similar architecture and tissue proliferation to those found in vivo. We show that cResponse is able to preserve human cancer tissue in 3D together with its microenvironment, including endothelial and immune cells, at a high viability (>90%) with continued cell division for 7 days in over twenty types of solids tumors. Importantly, the high viability of the tissue over an extended period of time allows for a rapid genomic profiling to help prioritize drugs to be tested by cResponse, as well as the capacity to evaluate the effects of slow acting drugs such as targeted therapy. To validate the capacity of the cResponse platform to predict patient response to cancer treatment, forty two patients with different cancer types receiving diverse chemotherapeutic and targeted agents were recruited to the study and their tumors were tested using cResponse prior to initiation of treatment. Cancer types included bladder, pancreatic, lung, colorectal, breast and sarcoma. After five days of treatment ex-vivo, the samples were fixed and designated a viability score (ranging from 0 to 100) based on an algorithm composed of a panel of histological and morphological markers. The cResponse score was compared to the clinical response to treatment of 27 patients for which this data is already available. The results demonstrated that cResponse could predict the patient's response with a specificity of 82% (9/11) and a sensitivity of 93.75% (15/16) when compared to the patient's imaging results. In the future, the integration of this platform in directing anti-cancer treatment may lead to better response rate of cancer patients to therapy. Citation Format: Seth Salpeter, Vered Bar, Sara Aharon, Luba Torovsky, Adi Zundelevich, Hamutal Shachar, Hagit Shapira, Nancy Gavert, Ravid Straussman, Shay Golan, Eli Rosenbaum, Talia Golan, Raanan Berger, Zohar Dotan, Dan Leibovici, Shani Breuer, Yakir Rotenberg, Aviad Zick, Ayala Hubert, Hovav Nechushtan, Guy Neev. A clinical trial of cResponse, a functional assay for cancer precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT209.

The human papillomavirus (HPV) test has proven to be efficient in triaging women with abnormal Pap findings in women with low cytological atypia, but there is no data about the accuracy for large loop excision of transformation zone in cases of recurrent atypia.To assess the clinical correlation between results of HPV typing and conization histology in women who had recurrent abnormal Pap test results with no colposcopy findings.Our retrospective cohort study included 138 women enrolled in the Maccabi Healthcare Services who had consecutive atypical Pap test results for 2 years in which no abnormal colposcopic findings were detected. These women had an HPV typing and then conization.Among the total study population (n=138), 71.7% had negative histology, 19.6% had ≤ cervical intraepithelial neoplasia grade 1 (≤ CIN1), and 8.7% had CIN2+. With regard to HPV typing, 34.8% were negative and 65.2% were positive. Of those testing positive, 34.4% were positive for HPV 16 or 18. Sensitivity, specificity, positive predictive value, and negative predictive values of HPV typing for women were 89.7%, 44.4%, 38.9%, and 91.7%, respectively, and for HPV 16 or 18: 71.4%, 67.7%, 32.3%, and 100.0%, respectively. After stratification by cytological grades, for women with high-grade cervical cytology, the sensitivity and negative predictive values of the HPV typing were higher than among low-grade cervical cytology, while specificity and positive predictive values were lower.HPV typing is a useful tool for the management of patients with persistently abnormal Pap test results.

OBJECTIVES This study aimed to assess the prevalence of human papillomavirus (HPV) in Israeli patients with cervical cancer and cervical intraepithelial neoplasia 3 (CIN3), to describe the distribution of the virus genotypes among positive cases, to characterize patients positive to HPV and, in particular, patients positive to HPV-16 and/or -18, and to evaluate the possible contribution of implementing HPV vaccination in Israel. METHODS Samples from 84 patients with cervical cancer and 886 patients with CIN3, archived at the Maccabi Institute of Pathology, were screened for HPV. DNA extraction was performed using DNeasy Blood and Tissue Kit/QIAGEN. HPV detection and typing were performed by multiplex polymerase chain reaction with primers E6/E7, using the f-HPV/Genomed kit. RESULTS Of the samples from 84 patients with cervical cancer, 89.3% were positive for HPV. Among these positive samples, HPV-16 was found in 70.7% and HPV-18 was found in 9.3%. Of the samples from 886 patients with CIN3, 85.0% were positive for HPV. Among these positive samples, HPV-16 was found in 73.8% and HPV-18 was found in 1.1%. In the patients with CIN3, the prevalence of HPV genotypes 16 and/or 18 was higher among young women and decreased across age groups. In addition, age, being born in Israel, being born in Europe, and being born in the former Soviet Union were correlated with a low risk of being infected with genotypes 16 and/or 18. DISCUSSION The prevalence of HPV-16 and -18 in patients with cervical cancer and CIN3 in Israel is high. It is expected that the implementation of routine vaccination against these types of HPV will significantly reduce the burden of these diseases in Israel.

We showed previously that proliferating human islet-derived de-differentiated cells (DIDs) exhibit many characteristics of mesenchymal stem cells. Dispersed DIDs can be induced by serum deprivation to undergo mesenchymal-to-epithelial transition and aggregate into epithelial cell clusters (ECCs). Conversely, ECCs can be induced to disperse and undergo epithelial-to-mesenchymal transition (EMT) by re-addition of mammalian sera. In this study, we show that platelet-derived growth factor BB (PDGF-BB) mimics and mediates serum-induced ECCs' dispersal accompanied by accumulation of cytoplasmic β-catenin and a decrease in the levels of insulin and glucagon mRNAs. Moreover, we show that PDGF-BB-induced dispersal of ECCs is a more general phenomenon that occurs also with bone marrow mesenchymal stem cells (BM-MSCs) and dermal fibroblasts (DFs). In DIDs, BM-MSCs, and DFs, PDGF decreased the levels of DKK1 mRNA, suggesting involvement of the Wnt signaling pathway. PDGF-BB stimulated a significant increase in S473 phosphorylation of Akt and the PI3K specific inhibitor (PIP828) partially inhibited PDGF-BB-induced ECC dispersal. Lastly, the PDGF-receptor (PDGF-R) antagonist JNJ-10198409 inhibited both PDGF-BB--and serum-induced ECC dispersal. Epidermal growth factor (EGF), which shares most of the PDGF signaling pathway, did not induce dispersal and only weakly stimulated Akt phosphorylation. Our data suggest that PDGF-BB mediates serum-induced DIDs dispersal, correlated with the activation of the PI3K-Akt pathway.

Purpose This study describes the distribution and the trends of cervical abnormalities in Israel, based on Pap smear results. Methods A retrospective analysis of cervical smears received by the Central Pathology Laboratory of Maccabi Healthcare Services between January 2005 and December 2010. Results In total, 711,541 Pap smears were screened in the study period. Cytological abnormalities were observed in 4.78 % of the total smears screened. An increase was observed in the rate of positive results from 2.63 % in 2005 to 6.78 % in 2010 (p = 0.0026). The cervical abnormalities in the study period distributed as follows: atypical squamous cell (ASC)—2.72 %, low-grade squamous intraepithelial lesion (LSIL)—1.54 %, high-grade squamous intraepithelial lesion (HSIL)—0.34 %, squamous cell carcinoma—0.01 %, atypical glandular cells (AGC)— 0.10 %, adenocarcinoma in situ (AIS)—0.06 % and invasive adenocarcinoma—0.01 %. The increase was statistically significant for ASC (p = 0.0028), LSIL (p = 0.0069) and for HSIL (p = 0.0260). The mean ages at diagnosis of women with ASCUS, LSIL, HSIL, squamous cell carcinoma, AGC, AIS and adenocarcinoma were 37.8, 33.2, 38.6, 55.4, 41.1, 49.9 and 57.1 years, respectively. Conclusions The increase in the rate of squamous cell abnormalities demonstrated in this study emphasizes the need of implementing an education and a screening program among Israeli women. HPV vaccine, sexual behavior, cytology performance and HPV test are primary and secondary prevention tools which may reduce morbidity and mortality in the future. In addition, based on the age at diagnosis of the different pathologies, the age group in which Pap test is performed in Israel should be expanded from 35–54 to 25–65 years.

High levels of plasminogen activator inhibitor-1 (PAI-1), which is produced by stromal, endothelial, and cancer cells and has multiple complex effects on cancers, correlate with poor cancer prognosis. To more definitively study the role of endogenously produced PAI-1 in human pancreatic adenocarcinoma (PAC) PANC-1 cell line biology, we used anti-PAI-1 shRNA to create stable PAI-1 deficient cells (PD-PANC-1s). PD-PANC-1s exhibited a heterogeneous morphology. While the majority of cells exhibited a cuboidal shape similar to the parental PANC-1 or the vector-infected control cells, numerous large cells with long filopodia and a neuronal-like appearance were observed. Although both Vector-control cells and PD-PANC-1s expressed mRNAs that are characteristic of mesenchymal, neural, and epithelial phenotypes, epithelial marker RNAs were up-regulated (e.g., E-cadherin, 32-fold) whereas mesenchymal marker RNAs were down-regulated (e.g., Thy1, ninefold) in PD-PANC-1s, suggesting mesenchymal-to-epithelial transition. Neural markers exhibited both up- and down-regulation. Immunocytochemistry indicated that epithelial-like PD-PANC-1s expressed E-cadherin and β-catenin in significantly more cells, while neural-like cells exhibited robust expression of organized β-3-tubulin. PAI-1 and E-cadherin were rarely co-expressed in the same cells. Indeed, examination of PAI-1 and E-cadherin mRNAs expression in additional cell lines yielded clear inverse correlation. Indeed, infection of Colo357 PAC cells (that exhibit high expression of E-cadherin) with PAI-1-expressing adenovirus led to a marked decrease in E-cadherin expression and to enhanced migration of cells from clusters. Our results suggest that endogenous PAI-1 suppresses expression of E-cadherin and differentiation in PAC cells in vitro, supporting its negative impact on tumor prognosis.

Native Xenopus oocytes exhibit dose-dependent depolarizing current responses to lysophosphatidic acid (LPA), with EC50 = 0.18 microM. Responses to LPA were subject to pronounced rapid desensitization. When oocytes were challenged with 5 nM LPA, the response was

This study describes time trends of cervical cancer and Cervical Intraepithelial Neoplasia 3 (CIN3) in Israel in the years 1986–2010 and characterizes these patients by demographics. A retrospective survey based on cervical cancer and CIN3 data documented in the computerized system of the second largest Health Maintenance Organizations (HMO) in Israel, “Maccabi Healthcare Services” (MHS) between 1986 and 2010. 737 cervical cancer patients and 3,459 patients of CIN3 were reported between 1986 and 2010. The mean age of women with cervical cancer was significantly higher (mean 49.1 years) than that of CIN3 patients (mean 36.3 years) (p-value

Xenopus oocytes respond to trypsin with a characteristic chloride current, virtually indistinguishable from responses mediated by a large number of native and expressed G protein-coupled receptors. We studied the involvement of G proteins of the Galphaq family as possible mediators of this and other G protein-coupled receptor-mediated responses in Xenopus oocytes. We have cloned the third member of the Galphaq family, Xenopus Galpha14, in addition to the previously cloned Xenopus Galphaq and Galpha11 (Shapira, H., Way, J., Lipinsky, D., Oron, Y., and Battey, J. F. (1994) FEBS Lett. 348, 89-92). Amphibian Galpha14 is 354 amino acids long and is 93% identical to its mammalian counterpart. Based on the Galpha14 cDNA sequence, we designed a specific antisense DNA oligonucleotide (antiGalpha14) that, together with antiGalphaq and antiGalpha11, was used in antisense depletion experiments. 24 h after injection into oocytes, either antiGalphaq or antiGalpha14 reduced the response to 1 microg/ml trypsin by 70%, whereas antiGalpha11 had no effect. A mixture of antiGalphaq and antiGalpha14 virtually abolished the response. These data strongly suggest that Galphaq and Galpha14 are the exclusive mediators of the trypsin-evoked response in Xenopus oocytes. Similar experiments with the expressed gastrin-releasing peptide receptor and muscarinic m1 receptor revealed the coupling of Galphaq and Galpha11, but not Galpha14, to these receptors in oocytes. These results confirm the hypothesis that endogenous members of the Galphaq family discriminate among different native receptors in vivo.

Proteinase-activated receptor-1 (PAR-1) and PAR-2 have been associated with increased invasiveness and metastasis in human malignancies. The role of PAR-3 has been less investigated. We examined the role of PARs in a human pancreatic adenocarcinoma PANC-1 cell line phenotype in vitro.We knocked down PAR-1, PAR-2, or PAR-3, whereas empty vector-infected cells served as controls. Specific peptide agonists of PARs were used to stimulate the receptors. In vitro assays of colony formation, migration, and invasion were used to characterize the phenotypes, and Western analysis was used to follow cell division control protein 42 homolog (CDC42) expression.PAR-1 and PAR-2 knockdowns (KDs) were markedly less, whereas PAR-3 KDs were robustly more migratory and invasive than the controls. Stimulation of PAR-1 or PAR-2 by their peptide agonists increased, whereas PAR-3 agonist reduced the invasion of the control cells. Knockdowns of all three PARs exhibited changes in the expression of CDC42, which correlated with the changes in their invasion. Conversely, stimulation of vector-control cells with PAR-1 or PAR-2 agonists enhanced, whereas PAR-3 agonist reduced the expression of CDC42. In the respective KD cells, the effects of the agonists were abrogated.The expression and/or activation of PARs is linked to the invasiveness of PANC-1 cells in vitro, probably via modulation of the expression of CDC42.

Agonist-induced calcium (Ca) mobilization is accompanied by Ca efflux, presumably reflecting the rise in Ca concentration at the cytosolic surface of the cell membrane. We studied the relationship between Ca efflux and intracellular Ca mobilization in Xenopus oocytes. Elevation of cytosolic Ca by a direct injection of 1 nmol 45 CaC1 2 resulted in a typical Ca-activated chloride current, but not in 45 Ca efflux. This demonstrated that a Ca rise at the cytoplasmic surface of the membrane is not sufficient to produce an increased efflux. Co-injection of inositol 1,4,5-trisphosphate (InsP 3 ), to prevent rapid Ca sequestration, also failed to cause Ca efflux. Smaller amounts of labelled Ca (0.05 nmol) equilibrated with Ca stores in a time-dependent pattern with an optimum at 2 h after injection. In contrast, Ca taken up from the medium was immediately available for agonist- or InsP 3 induced efflux. Emptying the agonist sensitive stores with thapsigargin (TG) did not affect chloride currents induced by Ca injection, indicating that these currents were due to direct elevation of Ca at the plasma membrane, rather than Ca-induced Ca release from InsP 3 sensitive stores. Agonist-induced depletion of Ca stores enhanced uptake from the extracellular medium and the subsequent release of the label by an agonist. Similar protocol when the label was injected into the oocytes, failed to affect agonist induced efflux. We suggest that, under physiological conditions, agonist-dependent Ca extrusion or uptake in oocytes is executed exclusively via a functionally restricted compartment, which is closely associated with both agonist-sensitive Ca stores and the plasma membrane.

G-proteins of the q family have been implicated as mediators of bombesin receptors action. We cloned Xenopus Gαq and Gα11 and specifically disrupted the synthesis of either protein with selective antisense oligonucleotides. Gαq antisense inhibited responses mediated by neuromedin B receptor (NMB-R) by 74%, though not by gastrin-releasing peptide receptor (GRP-R). Gα11 antisense had little effect on either GRP-R- or NMB-R-mediated responses. This suggests that NMB-R couples to Gαq, and that GRP-R and NMB-R show distinct G-protein coupling preferences in the Xenopus oocyte.

The two bombesin receptor subtypes, neuromedin B (NMB-R) and gastrin releasing peptide (GRP-R) receptors, bind their respective ligands with high affinity. To identify molecular components mediating high affinity NMB binding, four mutant receptors were constructed, in which different parts of the NMB-R were replaced with the corresponding regions of the GRP-R. When stably expressed in Balb 3T3 fibroblasts, all four NMB-R/GRP-R chimeras were functional and showed NMB-induced stimulation of inositol phosphate (IP) formation. Results of 125I-[D-Tyr0]NMB displacement assays using unlabeled NMB for competition indicated that high affinity NMB binding was determined by amino acid sequences in transmembrane domain V (TM-V) of the NMB-R. To identify which amino acid(s) in TM-V of NMB-R contributed to high affinity NMB binding, four additional NMB-R mutants were constructed where non-conserved amino acids in TM-V of NMB-R were replaced by the corresponding GRP-R amino acids. Three of the mutations, TyrPheLeu220-222-->PheTyrVal, Ile230-->Val, and His234-->Phe, did not affect high affinity NMB binding. The Ile216-->Ser substitution, however, abolished high affinity NMB binding and severely impaired the ability of the mutant receptor to stimulate NMB-dependent inositol phosphate formation. These results suggest that ILe216 in TM-V of NMB-R may be critical for high affinity NMB binding.

The bombesin (BN)-like peptides mediate a diverse spectrum of biological activities and have been implicated as autocrine growth factors in the pathogenesis and progression of some human small cell lung carcinoma tumors. Previously, two mammalian BN-like peptide receptor subtypes, gastrin-releasing peptide receptor and neuromedin-B receptor, have been cloned and characterized. In this study, we have isolated and characterized human genomic and complementary DNA (cDNA) clones encoding a new BN-like peptide receptor subtype, BN receptor subtype 3 (BRS-3). Expression of BRS-3 cDNA in Xenopus oocytes encodes a functional receptor that is specifically activated by BN-like peptides. Chromosome mapping studies indicate that the BRS-3 gene is located on human chromosome X. BRS-3 mRNA expression in rat tissues is limited to secondary spermatocytes in testis. In contrast, BRS-3 mRNA is widely expressed in a panel of human cell lines from all histological types of lung carcinoma. These results suggest a role for BN-like peptides and their receptors in mammalian reproductive physiology and also indicate that BRS-3 could serve as a potential therapeutic target for human lung carcinoma.

The cholecystokinin (CCK) family of peptides and their receptors are widely distributed throughout the gastrointestinal and central nervous systems where they regulate secretion, motility, growth, anxiety, and satiety. The CCK receptors can be subdivided into at least two subtypes, CCKA and CCKB on the basis of pharmacological studies. We report here the purification of the CCKA receptor to homogeneity from rat pancreas by using ion-exchange and multiple affinity chromatographic separations. This allowed partial peptide sequencing after chemical/enzymatic cleavage and synthesis of degenerate oligonucleotide primers. These primers were used for initial cloning of the cDNA from rat pancreas by PCR. The predicted protein sequence of the cDNA clone contained the five partial peptide sequences obtained from the purified protein. Seven putative transmembrane domains suggest its membership in the guanine nucleotide-binding regulatory protein-coupled receptor superfamily. In vitro transcripts of the cDNA clone were functionally expressed in Xenopus oocytes and displayed the expected agonist and antagonist specificity.

Bombesin-like peptides have been implicated as autocrine growth factors influencing the pathogenesis and progression of some human lung carcinoma cells. To determine the pharmacologic and structural properties of the bombesin receptors expressed in human lung carcinoma cells, cDNA clones encoding a human gastrin-releasing peptide receptor (GRP-R) and a pharmacologically distinct neuromedin-B preferring bombesin-receptor (NMB-R) were isolated from a human small cell lung carcinoma cell line (NCI-H345). After expression in Xenopus oocytes, a GRP-R-specific antagonist was effective in blocking responses elicited from the cloned GRP-R, but not the NMB-R. Both GRP-R and NMB-R mRNA expression was detected at varying levels in a panel of human lung cancer cell lines. These results indicate heterogeneity of bombesin receptor subtypes exists in human lung carcinoma cells and should be considered in the design of bombesin receptor antagonists intended to inhibit tumor cell growth.

Physiological responses to mammalian bombesin-like peptides were studied in Xenopus oocytes injected with mRNA isolated from Swiss 3T3 cells and rat esophagus in order to identify and characterize bombesin receptor subtypes. Both groups respond similarly to either gastrin releasing peptide or neuromedin B, but only the response to neuromedin B in oocytes expressing the esophagus mRNA is not blocked by a specific gastrin releasing peptide receptor antagonist, des-Met-[D-Phe6]Bn(6-13) ethyl ester. Complete desensitization of gastrin releasing peptide-evoked responses in oocytes expressing esophagus mRNA does not abolish neuromedin B-evoked responses. A single application of neuromedin B abolishes responses to subsequently applied gastrin releasing peptide in oocytes expressing esophagus, but not Swiss 3T3, mRNA. RNA blot hybridization studies using a Swiss 3T3 gastrin releasing peptide receptor cDNA probe show no detectable hybridization in esophagus mRNA samples. These data suggest that a gastrin releasing peptide receptor is expressed in the esophagus and that it is distinct from that expressed in Swiss 3T3 cells and may represent a third subtype of mammalian bombesin receptor.

Recent binding studies in the central nervous system and other tissues provide evidence that the mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-B (NMB), exert their numerous physiological effects through at least two different receptors. We describe the structure and expression of a cloned NMB-preferring bombesin receptor (NMB-R) with properties distinct from a GRP-preferring bombesin receptor (GRP-R) reported previously. In particular, the NMB-R shows higher affinity binding to NMB than to GRP in BALB 3T3 fibroblasts expressing the cloned NMB-R. The distinct regional distribution of NMB-R and GRP-R mRNA in the brain suggests that both bombesin receptor subtypes play independent roles in mediating many of the dramatic effects of bombesin-like peptides in the central nervous system.

The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor is a member of the guanine nucleotide binding protein-coupled receptor superfamily with seven predicted hydrophobic transmembrane domains. In vitro transcripts from cloned cDNA templates encompassing the predicted protein coding domain, when injected into Xenopus oocytes, resulted in expression of functional GRP receptors. The predicted amino acid sequence of the open reading frame in cDNA clones matches the amino-terminal sequence as well as the sequence of four tryptic fragments isolated from the purified protein. Expression of the GRP receptor cDNA in model systems potentially provides a powerful assay for the development of subtype-specific receptor antagonists that may prove to be of therapeutic importance in human small cell lung carcinoma.

Oocytes of 40% of Xenopus laevis frogs respond to acetylcholine (ACh). Oocytes of the majority of responders exhibit the common two-component depolarizing muscarinic response (mean amplitude of the rapid component, 54 nA). Oocytes of approximately 10% of the responders ("variant" donors) exhibit a muscarinic response characterized by a very large transient, rapid current (mean amplitude 1242 nA, reversal potential -33 mV). Responses in oocytes of variant donors exhibit further qualitative differences: pronounced desensitization (absent in oocytes of common donors), characteristic prolonged latency (5.4 vs 0.9 s in oocytes of common donors) and marked inhibition of the response by activators of protein kinase C. Rapid responses in oocytes of variant donors are usually increased by treatment with collagenase, which, in common oocytes, often results in a complete loss of the response that correlates with the loss of muscarinic ligand binding. The number of muscarinic receptors was similar in oocytes of both types of donors (2.2 vs 3.0 fmol/oocyte). Also, the responses of oocytes of variant donors to microinjections of CaCl2 or inositol 1,4,5-trisphosphate were similar to those found in cells of common donors. These findings imply that altered receptor number, calcium stores and/or chloride channel density are not responsible for the variant responses. However, ACh caused an sixteen-fold greater efflux of 45Ca in oocytes of variant donors (35 vs 2.2% of total label in oocytes of common donors). Hence, the characteristics of the variant response may be related to a more efficient coupling between receptor stimulation and the mobilization of cellular calcium.

Acetylcholine (ACh) and thyrotropin-releasing hormone (TRH) utilize inositol 1,4,5-trisphosphate (IP3) as a second messenger and evoke independent depolarizing membrane electrical responses accompanied by characteristic 45Ca efflux profiles in Xenopus laevis oocytes injected with GH3 pituitary cell mRNA. To determine whether this could be accounted for by mobilization of calcium from functionally separate stores, we measured simultaneously 45Ca efflux and membrane electrical responses to ACh and TRH in single oocytes. We found that depletion of ACh-sensitive calcium store did not affect the membrane electrical response to TRH and the TRH-evoked 45Ca efflux. Our data suggest that ACh and TRH mobilize calcium from distinct cellular stores in the oocyte. This is the first demonstration in a single cell of strict subcellular compartmentalization of calcium stores coupled to two different populations of cell membrane receptors that utilize the same second messenger.

The mammalian bombesin (Bn)-like neuropeptide receptors gastrin-releasing peptide receptor (GRP-R) and neuromedin B receptor (NMB-R) transduce a variety of physiological signals that regulate secretion, growth, muscle contraction, chemotaxis and neuromodulation. We have used reverse transcription-polymerase chain reaction (PCR) to isolate a cDNA from human brain mRNA, GPCR/CNS, that encodes a putative G protein-coupled receptor (GPCR) based upon the presence of the paradigmatic seven heptahelical transmembrane domains in its predicted amino acid sequence. Analysis of the deduced protein sequence of GPCR/CNS reveals this putative receptor to be 98% identical to the deduced amino acid sequence of a recently reported gene product and minimally identical (approximately 23%) to both murine GRP-R and human endothelin-B (ET-B) receptor. Our deduced protein sequence differs at 12 positions, scattered throughout the open reading frame, relative to the original sequence. A 3.7 kb GPCR/CNS mRNA species is expressed in vivo in a tissue-specific manner, with highest levels detected in brain and spinal cord, lower levels found in testis, placenta and liver, but no detectable expression observed in any other tissue. Analysis of GPCR/CNS genomic clones reveals that the human gene contains one intron that is about 21 kb in length that divides the coding region into two exons and maps to human chromosome 7q31. No specific binding is observed with either a newly identified ligand (DTyr6, beta Ala11, Phe13, Nle14]Bn-(6-14)) having high affinity for all Bn receptor subtypes or Bn after GPCR/CNS is stably expressed in fibroblasts. No elevation in inositol trisphosphate is observed after the application of micromolar levels of either DPhe6, beta Ala11, Phe13, Nle14]Bn-(6-14) or Bn, a concentration of agonist known to activate all four known Bn receptor subtypes. When GPCR/CNS is expressed in Xenopus oocytes, no activation of the calcium-dependent chloride channel is detected despite the addition of micromolar levels of Bn peptide agonists. We conclude that the natural ligand for this receptor is none of the known naturally occurring Bn-like peptides and the true agonist for GPCR/CNS remains to be elucidated.

Pseudohypoparathyroidism type Ia (PHP-Ia) is a hereditary disease characterized by resistance to PTH and other hormones that act via cAMP. Patients have deficient activity of Gs alpha, the alpha subunit of the G protein, which couples hormone receptors to stimulation of adenylate cyclase. We describe two new mutations discovered in two sporadic patients with PHP-Ia. Using genomic DNA, we have amplified exons 2-13 of the Gs alpha gene (GNAS1) by PCR, and sequenced the resulting products. Both patients had Albright's hereditary osteodystrophy, resistance to multiple hormones, and deficient Gs alpha activity. In the first patient, a deletion of a C in exon 5 at codon 115 was found. In the second patient, an insertion of a C in exon 10 at codon 267 was detected. Both these heterozygous mutations cause frameshift, and predict decreased production of Gs alpha. This report adds two new Gs alpha mutations to the known ten mutations recently described.

Publisher Summary Bombesin (BN) is a tetradecapeptide purified from the skin of the European frog, Bombina bombina. Many BN-like peptides have been isolated from various amphibians. These peptides have similar carboxy-terminal heptapeptide sequences. They are classified into three subfamilies based on the last three amino acids of their α-amidated carboxy termini: the BN subfamily (His-Leu-Met-NH2), the ranatensin subfamily (His-Phe-Met-NH2), and the phyllolitorin subfamily (Ser-Leu or Phe-Met-NH2). Two mammalian BN-like peptides have been described, gastrin-releasing peptide (GRP) in the BN subfamily and neuromedin B (NMB) in the ranatensin subfamily. Two distinct mammalian BN receptor subtypes have been identified, cloned, and characterized. The two receptors have different relative affinities for GRP and NMB. The GRP-preferring receptor (GRP-R) is expressed in pancreatic acinar cells and Swiss 3T3 fibroblasts, whereas a NMB-preferring receptor (NMB-R) is found in esophagus and C6 glioma cells. GRP-R and NMB-R are expressed in the central nervous system. This chapter describes the methods used for discriminating BN receptor subtypes. The two BN receptor subtypes (GRP-R and NMB-R) are distinguished by differences in their ligand binding properties in tissues and cultured cells. Isolation of cDNA clones for the two receptors provides additional possibilities for studying differences between GRP-R and NMB-R.

Angiotensin II (AII) is a major regulator of cardiovascular function and fluid homeostasis. Recently, the cDNA for an AII receptor (AT1) was cloned from rat smooth muscle and bovine adrenal. To search for AII receptor subtypes, we amplified rat adrenal cortex cDNA by PCR using primers based on the AT1 receptor. The product was distinct from the AT1 receptor as indicated by restriction enzyme analysis and DNA sequencing. A full-length cDNA clone (2.2 kilobase pairs) encoding a novel AII receptor (AT3) was obtained by screening an adrenal cortex library. The AT3 cDNA encodes a Mr 40,959 protein with 95% amino acid identity to the rat smooth muscle receptor, but the overall nucleotide similarity is 71% due to low homology in the 5'- (58%) and 3'- (62%) untranslated regions. Expressed AT3 receptors in Xenopus oocytes and COS-7 cells mediate agonist-induced Ca2+ mobilization but are pharmacologically distinct from the AT1 receptors. AT3 mRNA is most abundant in the adrenal cortex and pituitary and differs from AT1 mRNA in its tissue distribution. The structural features of the AT3 receptor, including two additional potential phosphorylation sites for protein kinase C, could be related to the distinctive binding properties of the adrenal and vascular receptors and to their differential regulation during altered sodium intake.

We tested the contribution of extracellular calcium (Ca o 2+ ) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Ca o caused a decrease in both the rapid (D 1 ) and the slow (D 2 ) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca o 2+ removal on the muscarinic response was mimicked by the addition of 1 mM Mn 2+ , an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca 2+ -free medium, subsequent addition of 1.8 mM CaCl 2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca o 2+ -dependent component of membrane electrical responses, the Ca 2+ -evoked current was reversibly abolished by Mn 2+ , though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca 2+ -evoked current, implying negative feedback of calcium channels by calcium. Injection of 10–100 fmol ofinositol 1,4,5-trisphosphate (IP 3 ) resulted in a two-component depolarizing current. IP 3 injection promoted the appearance of Ca o 2+ -evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage- insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP 3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.

Muscarinic stimulation of follicle-enclosed oocytes ofXenopus laevis results in a complex response that involves both depolarizing and hyperpolarizing currents (Dascal and Landau 1980). We studied the involvement of protein kinase C (PK-C1) in the regulation of the acetylcholine-evoked rapid (D1) and of the slow (D2) depolarizing chloride (Cl−) currents. In oocytes maintained at −100 mV [the reversal potential of potassium (K+) ions] under two electrode voltage clamp, the PK-C activator 4-β-phorbol 12-myristate 13-acetate (β-PMA, 0.1 μM) stimulated D1 by 99±17% and inhibited D2 by 67±6%, vs. untreated controls. The inactive isomer (α-PMA) or phorbol alone had no significant effect on the components of the muscarinic response. In order to identify the site of the regulation, we have microinjected the intracellular second messenger of calcium mobilization, inositol 1,4,5-trisphosphate (IP3). β-PMA or the diacylglycerol analog, oleoylacetylglycerol (OAG) stimulated the rapid depolarizing current evoked by IP3 by 220±26% and 394±102%, respectively. α-PMA had little if any effect. The calcium-evoked Cl− current in oocytes pre-treated with the divalent cation ionophore A23187 was, on the other hand, inhibited by β-PMA and OAG (by 82±6% and 54±6%, respectively). α-PMA and phorbol had a limited inhibitory effect. β-PMA, but not α-PMA, also mildly inhibited the IP3-evoked increase in45Ca efflux. The intracellular metabolism of IP3 was not affected by exposure to either β-PMA or OAG. In conclusion, PK-C appears to regulate the acetylcholine-evoked Cl− response in a complex pattern: inhibition of the slow (D2) Cl− current (possibly directly on the Cl− channel) and stimulation of the rapid (D1) Cl− current. Both sites of regulation seem to be distal to IP3 metabolism and to IP3-evoked calcium mobilization. Our results are consistent with the possibility that the complex muscarinic response in Xenopus oocyte is mediated by two populations of Cl− channels.

Threshold concentrations of lysophosphatidic acid (LPA) or acetylcholine (ACh) induce pertussis toxin (PTX)-sensitive rapid desensitization of responses to LPA in Xenopus oocytes. To demonstrate that threshold [LPA] rapidly activates Gi/o proteins, we used the G protein-activated K+ channel (GIRK) as a reporter. Low [LPA] induced IK+ in

Shallow injection of inositol 1,4,5-trisphosphate (IP3) near the animal pole of the Xenopus oocyte resulted in a large depolarizing current that decayed rapidly. A similar injection near the vegetal pole produced a much smaller response characterized by a significantly slower rate of decay. Injection of CaCl2 near the animal pole of the oocyte resulted in a large depolarizing current characterized by rapid rise and decay times. Injection near the vegetal pole of the cell produced responses that exhibited similar amplitudes but much longer rise and decay times. The protein kinase C (PK-C) activator, β-phorbol 12-myristate 13-acetate (PMA), significantly enhanced the rapid responses to IP3 injections at either hemisphere but did not affect the amplitudes of the responses to CaCl2. The PK-C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) had no effect on the responses to CaCl2. These results imply an asymmetric distribution of calcium stores and chloride channels between the two hemispheres of the oocyte.