Cholera toxin inhibits signal transduction by several mitogens and the in vitro growth of human small-cell lung cancer.

Cholera toxin (CT) inhibited the in vitro growth of three of four human small-cell lung carcinoma (SCLC) cell lines with a 50% inhibitory concentration of 27-242 ng/ml. Loss of surface membrane ruffling and the capacity of [Tyr4]-bombesin, vasopressin, and fetal calf serum to stimulate increases in intracellular free calcium clearly preceded effects on cellular metabolic activity and cell growth. 125I-[Tyr4]-bombesin binding was unaffected by CT treatment but [Tyr4]-bombesin stimulated phospholipase C activity was decreased in membranes from CT-treated SCLC cells. CT stimulated a rapid but transient increase in intracellular cyclic AMP ([cAMP]i) in SCLC. The effects of CT on susceptible SCLC were not reproduced by elevations of [cAMP]i induced by forskolin or cyclic AMP analogues. GM1 ganglioside, the cellular binding site for CT, was highly expressed in the CT-sensitive but not the CT-resistant SCLC cell lines. In contrast, expression of guanine nucleotide binding protein substrates for ADP-ribosylation by CT was similar. These data demonstrate the existence of a CT-sensitive growth inhibitory pathway in SCLC-bearing GM1 ganglioside. Addition of CT results in decreased responsiveness to several mitogenic stimuli. These results suggest novel therapeutic approaches to human SCLC.

Unique catalytic properties dictate the enhanced function of p59fynT, the hemopoietic cell-specific isoform of the Fyn tyrosine protein kinase, in T cells.

As a result of alternative splicing, the fyn gene encodes two different tyrosine protein kinase isoforms. While one protein (p59fynB) is abundantly expressed in the brain, the alternative product (p59fynT) is contained only in cells of hemopoietic lineages, especially T lymphocytes. Sequence analyses have revealed that these two isoforms differ exclusively within a stretch of 52 amino acids which overlaps the end of the Src homology 2 (SH2) motif and the beginning of the catalytic domain. Consistent with the idea that FynT provides a specialized function in hemopoietic cells, we have previously shown that expression of activated FynT molecules, but not that of activated FynB polypeptides, enhanced the antigen responsiveness of a mouse T-cell line (BI-141) (D. Davidson, L. M. L. Chow, M. Fournel, and A. Veillette, J. Exp. Med. 175:1483-1492, 1992). In this study, we examined the basis for the distinct signalling capabilities of the two Fyn isoforms in T lymphocytes. Our biochemical analyses revealed that FynT is more adept than FynB at promoting antigen receptor-triggered calcium fluxes. This phenomenon likely contributes to the improved biological function of FynT during antigen stimulation, as the calcium ionophore ionomycin partially rescued the inability of FynB to enhance antigen-induced lymphokine secretion. To establish the structural basis for these observations, we also created and analyzed a series of chimeras of FynT and FynB. These studies demonstrated that the distinct catalytic domain of FynT, and not its altered SH2 motif, is responsible for the improved ability to augment antigen responsiveness. Similarly, this sequence enhances the ability to mobilize cytosolic calcium in response to antigen receptor stimulation. Taken together, these data show that the distinct biological impacts of FynT and FynB in T cells are related to limited structural differences in the amino-terminal portion of their catalytic domains and that they reflect, at least in part, the greater ability of FynT to mobilize cytoplasmic calcium.

Receptor subtype expression and responsiveness to bombesin in cultured human bronchial epithelial cells.

We detected low level expression of the gastrin-releasing peptide and neuromedin-B receptor mRNAs in cultures of human bronchial epithelium from 4 of 6 individuals. Bombesin receptor subtype-3 mRNA was undetectable in these cells. An elevation of intracellular calcium concentration was observed in response to bradykinin (6 of 6) and neurotensin (1 of 5) but not to bombesin (0 of 6), vasopressin (0 of 6), or cholecystokinin (0 of 3). In contrast, such responses are frequently noted in lung cancer cell lines. Bombesin did not stimulate the in vitro growth of an immortalized human bronchial epithelium cell line expressing low levels of bombesin receptor mRNAs. We conclude that bombesin receptors are expressed at low levels in human bronchial epithelium cells which may acquire greater responsiveness to multiple neuropeptides in the course of multistep carcinogenesis.

Expression of the gastrin-releasing peptide receptor confers a growth response to bombesin in immortalized human bronchial epithelial cells.

We have introduced a human gastrin-releasing peptide receptor expression vector into an immortalized human bronchial epithelial cell normally unresponsive to the ligand bombesin. Successfully transfected cells express specific binding sites at a density similar to that found at the surface of human lung cancer cells and show an elevation of intracellular calcium concentration in response to bombesin. We found that cellular strains expressing the receptor showed a growth stimulation in response to bombesin in proportion to cell surface receptor density. We conclude that expression of bombesin receptors contributes to the growth potential of human bronchial epithelial cells.

Cholera toxin triggers apoptosis in human lung cancer cell lines.

Cholera toxin (ChT) inhibits signals generated by multiple growth factors in human lung cancer cells, resulting in cell growth inhibition. We now report that ChT triggers apoptosis as shown by DNA fragmentation and activation of caspases cleaving poly(ADP-ribose) polymerase and lamin B. Apoptosis induced by ChT in a small cell lung cancer cell line is not affected by manipulations of intracellular cAMP through preincubation with isobutylmethylxanthine but can be modestly increased through inhibition of protein kinase C with chelerythrine. Thus, apoptosis is actively suppressed in lung cancer cells by a ChT-sensitive-growth regulatory pathway, and these observations may have significant implications in the development of novel strategies for lung cancer treatment.

Effect of guanine and adenine nucleotides on bombesin-stimulated phospholipase C activity in membranes from Swiss 3T3 and small cell lung carcinoma cells.

In [3H]inositol-labeled membranes prepared from Swiss mouse 3T3 and human small cell lung carcinoma cells, [Tyr4]-bombesin stimulated production of water-soluble inositol phosphates. The reaction was stimulated by guanosine 5'-O-[3-thiotriphosphate] and was specifically inhibited by both [Leu13-psi-CH2NHLeu14]-bombesin and the antibombesin antibody 2A11. [Tyr4]-bombesin-induced activation of phospholipase C is most apparent in Ca2(+)-depleted conditions (less than 1 microM[Ca2+]free). The kinetics of activation by ligand also demonstrate that [Tyr4]-bombesin-dependent phospholipase C activation is most apparent at [Mg2+]free of approximately 0.2 microM. At millimolar concentrations of [Mg2+]free, there is considerably less dependence on [Tyr4]-bombesin for activation of phospholipase C. ATP is not necessary for initial activation of phospholipase C, and beta, gamma-imidoadenosine-5'-triphosphate does not inhibit the reaction. These results demonstrate that in these cell types [Tyr4]-bombesin activates phospholipase C in conjunction with guanine nucleotides. Phospholipase C-coupled guanine nucleotide regulatory proteins would be appropriately considered as novel targets for the development of therapeutic strategies in small cell lung carcinoma.

Elevated DNA repair capacity is associated with intrinsic resistance of lung cancer to chemotherapy.

Non-small cell lung cancer (N-SCLC) is generally unresponsive to chemotherapy even without previous drug treatment, as opposed to small cell lung cancer (SCLC), which is initially responsive to chemotherapy. The mechanisms of this intrinsic resistance are unknown. This study was designed to investigate the role of DNA repair in intrinsic resistance of N-SCLC to cisplatin. A panel of primary N-SCLC cell cultures and established cell lines were examined and compared to SCLC cell lines established previously from untreated patients. The overall DNA repair capacity was estimated by the ability of cells to reactivate the pRSV-CAT plasmid damaged by cisplatin ("host cell reactivation" assay). Cytotoxicity was determined for cisplatin in vitro. N-SCLC cells were found to be significantly more resistant to cisplatin than SCLC cell lines isolated from untreated patients (P < 0.01). The capacity of N-SCLC cells to reactivate pRSV-CAT plasmid damaged with cisplatin and transfected into cells was higher in N-SCLC cells than in SCLC cells originating from patients who were untreated previously (P < 0.05). Correlation was also observed between chloramphenicol acetyltransferase activity and intrinsic resistance to cisplatin. However, no significant difference was observed between primary N-SCLC cultures and established cell lines. This study indicates that elevated DNA repair capacity is associated with drug resistance in lung cancer and suggests that modulation of DNA repair mechanism(s), such as the incorporation of specific DNA repair inhibitor(s) in therapeutic regimens, may help to improve therapeutic strategies of N-SCLC.

Growth inhibition by cholera toxin of human lung carcinoma cell lines: correlation with GM1 ganglioside expression.

The effect of cholera toxin (CT) on the growth of 12 small cell lung carcinoma (SCLC) and 15 non-small cell lung carcinoma (NSCLC) cell lines is presented. CT inhibited the growth of nine SCLC cell lines (concentration for 50% inhibition of growth, 27-700 ng/ml), all of which had abundant expression of GM1 ganglioside, the surface receptor for CT. CT-resistant SCLC all had greatly decreased GM1 expression. In contrast, CT inhibited the growth of only four of 15 NSCLC cell lines. Seven of the 11 CT-resistant NSCLC had levels of GM1 comparable to CT-sensitive NSCLC or SCLC. In a limited panel of cell lines, cyclic AMP (cAMP) agonists including forskolin, 8Br[cAMP], and dibutyryl[cAMP] did not consistently reproduce CT-mediated inhibition of cell growth, nor did these compounds overcome resistance of cells to the growth inhibitory effects of CT. Expression of the RI and RII regulatory subunits of cAMP-dependent protein kinase was similar in CT-resistant and CT-sensitive SCLC or NSCLC cell lines. In the presence of isobutylmethylxanthine, intracellular cAMP levels induced by CT in a CT-resistant, GM1(+) NSCLC cell line were comparable to those achieved in a CT-sensitive NSCLC cell line. We conclude that inhibition of lung carcinoma cell growth by CT in all cases requires expression of GM1, and in the case of SCLC cell lines the presence of GM1 is sufficient. In NSCLC cell lines, expression of GM1 is not sufficient for growth inhibition by CT. These findings imply refractoriness to growth inhibition by cAMP in GM1(+), CT-resistant NSCLC cell lines and the possibility of non-cAMP-related mechanisms for growth inhibition in CT-sensitive cell lines.

ras gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent.

We analyzed 66 non-small cell lung cancer cell lines for mutations at codons 12, 13, and 61 of all three ras genes and correlated the findings with patient survival. We used designed restriction fragment-length polymorphisms to detect mutations after amplification of ras-specific sequences by the polymerase chain reaction. We found 19 mutations of ras genes (29%), and 11 of these 19 (58%) were at codon 12 of the K-ras gene. By univariate analysis, the presence of any ras mutation in cell lines from patients who received curative intent treatment was associated with a shorter survival (P2 = 0.002). For patients who received only palliative treatment, detection of K-ras mutations at codon 12 was associated with a shortened survival (P2 = 0.0103), but this analysis was not statistically significant for the group with any ras mutation (P2 = 0.093). The Cox proportional hazards model also predicted a higher risk for patients with any type of ras mutations. We conclude that ras mutations, present in a subset of non-small cell lung cancers, are independently associated with the shortened survival of patients, irrespective of treatment intent.

Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines.

We screened a panel of 103 human lung cancer cell lines for the presence of point mutations at codons 12, 13 or 61 of the human K-, H- and N-ras genes, using restriction fragment length polymorphisms (RFLP), created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA. We found ras mutations in 22/61 (36%) non-small-cell lung cancer (NSCLC) cell lines, predominantly in K-ras codon 12. Identical mutations were present in uncultured tumor materials corresponding to 11 cell lines containing mutated ras genes. ras mutations were found not only in adenocarcinoma cell lines (9/32, 28%), but also in cell lines derived from other types of NSCLC (13/29, 45%). In contrast, none of 37 small-cell lung cancer (SCLC) cell lines and five extra-pulmonary small-cell cancer cell lines had ras mutations. ras mutations were not correlated with sex of the patients, tumor extent, prior therapy status or in vitro culture time. G to T or A to T transversions were the most common base substitutions, occurring in codons 12 and 61 respectively. We conclude that ras mutations play a role in the pathogenesis of a subset of NSCLC but are not involved in SCLC.

Tirapazamine with cisplatin in patients with advanced non-small-cell lung cancer: a phase II study.

PURPOSE: A phase II study was conducted to evaluate the safety and efficacy of tirapazamine combined with cisplatin for the treatment of patients with advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Forty-four patients with stage IIIB/IV NSCLC were treated with a combination of tirapazamine and cisplatin. Patients received tirapazamine 260 mg/m2 administered intravenously over 2 hours, followed 1 hour later by cisplatin 75 mg/m2 administered over an additional hour, repeated every 21 days. The duration of therapy was meant to be limited to four cycles for nonresponders and eight cycles for responders. RESULTS: Ten of 44 patients (23%) showed a partial response. The estimated median survival for all patients was 37 weeks. Toxicities were treatable and included grade 3 nausea or vomiting (25%), fatigue (27.3%), and muscle cramps (4.5%). No dose reductions were necessary. CONCLUSION: The results show that tirapazamine can safely be added to cisplatin. Both the median survival and response rate observed strongly suggest that tirapazamine with cisplatin is more active than cisplatin alone.

Tirapazamine plus cisplatin versus cisplatin in advanced non-small-cell lung cancer: A report of the international CATAPULT I study group. Cisplatin and Tirapazamine in Subjects with Advanced Previously Untreated Non-Small-Cell Lung Tumors.

PURPOSE: A phase III trial, Cisplatin and Tirapazamine in Subjects with Advanced Previously Untreated Non-Small-Cell Lung Tumors (CATAPULT I), was designed to determine the efficacy and safety of tirapazamine plus cisplatin for the treatment of non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with previously untreated NSCLC were randomized to receive either tirapazamine (390 mg/m(2) infused over 2 hours) followed 1 hour later by cisplatin (75 mg/m(2) over 1 hour) or 75 mg/m(2) of cisplatin alone, every 3 weeks for a maximum of eight cycles. RESULTS: A total of 446 patients with NSCLC (17% with stage IIIB disease and pleural effusions; 83% with stage IV disease) were entered onto the study. Karnofsky performance status (KPS) was >/= 60 for all patients (for 10%, KPS = 60; for 90%, KPS = 70 to 100). Sixty patients (14%) had clinically stable brain metastases. The median survival was significantly longer (34.6 v 27. 7 weeks; P =.0078) and the response rate was significantly greater (27.5% v 13.7%; P <.001) for patients who received tirapazamine plus cisplatin (n = 218) than for those who received cisplatin alone (n = 219). The tirapazamine-plus-cisplatin regimen was associated with mild to moderate adverse events, including acute, reversible hearing loss, reversible, intermittent muscle cramping, diarrhea, skin rash, nausea, and vomiting. There were no incremental increases in myelosuppression, peripheral neuropathy, or renal, hepatic, or cardiac toxicity and no deaths related to tirapazamine. CONCLUSION: The CATAPULT I study shows that tirapazamine enhances the activity of cisplatin in patients with advanced NSCLC and confirms that hypoxia is an exploitable therapeutic target in human malignancies.

Chick Delta-1 gene expression and the formation of the feather primordia.

The chick dermis is known to control the formation of feathers and interfeathery skin in a hexagonal pattern. The evidence that the segregation of two types of fibroblasts involves Delta/Notch signalling is based on three facts. Rings of C-Delta-1-expressing fibroblasts precede and delimit the forming feather primordia. C-Delta-1 is uniformly expressed in the dermis of the scaleless mutant, which is almost entirely devoid of feathers. Feather development is inhibited by overexpression of C-Delta-1 in wild type dermis using a retroviral construct. We also show that the distribution of C-Delta-1 in the mutant dermis can be rescued by its association with a wild type epidermis, which acts as a permissive inducer, or by epidermal secreted proteins like FGF2.

Angiogenesis correlates with vascular endothelial growth factor expression but not with Ki-ras oncogene activation in non-small cell lung carcinoma.

A total of 195 non-small cell lung carcinoma (NSCLC) specimens were studied for the presence of mutations in their ras family genes, for tumor vascularity, and for their immunostaining pattern with an antibody to vascular endothelial growth factor (VEGF). ras mutation was found in 37 of 104 (34.6%) adenocarcinoma specimens, in 0 of 64 squamous cell carcinomas, and in 2 of 27 (7.4%) large cell undifferentiated carcinomas. All mutations were found on the Ki-ras gene, with 37 (95%) of them on codon 12 and the remaining 2 on codon 13. Thirty (77%) of the mutations were G to T transversions. There was a correlation between increasing tumor vascularity and VEGF immunostaining score, but there was no correlation between either of them with the activation of the ras oncogene. A study of VEGF mRNA expression in 14 NSCLC cell lines also demonstrated a lack of correlation between the constitutive expression levels of VEGF and the presence or absence of ras mutation in these cell lines. The results suggest that VEGF is a major angiogenesis factor in NSCLC but that other factors beside ras mutations may influence tumor vascularity in these tumors. The two parameters may potentially serve as independent prognostic factors in NSCLC.

Differential expression of two different homeobox gene families during mouse tegument morphogenesis.

The expression of six genes belonging to two different homeobox gene families was studied during the embryonic and postnatal morphogenesis of head and body regions of the mouse integument. The first family included the Otx1 and Otx2 genes, both related to the orthodenticle Drosophila gene and the second was represented by four members of the Antennapedia class HOX genes: Hoxc8 and three Hoxd genes, d9, d11 and d13. In situ hybridizations with 35S labeled antisense RNA probes were performed on head serial frontonasal sections, as well as entire embryo and postnatal tail longitudinal sections. The expression of these genes shows a differential spatiotemporal pattern along the cephalo-caudal axis. In 12.5-day and 15.5-day embryos, the Otx2 gene expression is restricted to the nasal epithelium and its associated glands, while the Otx1 transcripts are present in both nasal and facial integuments, including nasal glands and hair vibrissa follicles. The Hoxc8 expression first appears in skin at 14.5 days of gestation in the sternal region and is extended at 16.5 days to the thoracic ventral and lumbar dorsal regions. The Hoxd9 and Hoxd11 genes are only expressed in the caudal skin from 14.5 days of gestation. The Hoxd13 transcripts are the last to appear, 2 days after birth, and are limited to the last epidermal cells to differentiate, i.e. those of the hair matrix of the caudal pelage hair follicles. Taken together, these observations strengthen the hypothesis that different homeobox gene families specify the regional identity of the skin in the cephalic and body regions.

Androgen-regulated microRNA-135a decreases prostate cancer cell migration and invasion through downregulating ROCK1 and ROCK2.

Androgen signaling, via the androgen receptor (AR), is crucial in mediating prostate cancer (PCa) initiation and progression. Identifying new downstream effectors of the androgens/AR pathway will allow a better understanding of these mechanisms and could reveal novel biomarkers and/or therapeutic agents to improve the rate of patient survival. We compared the microRNA expression profiles in androgen-sensitive LNCaP cells stimulated or not with 1 nM R1881 by performing a high-throughput reverse transcriptase-quantitative PCR and found that miR-135a was upregulated. After androgen stimulation, we showed that AR directly activates the transcription of miR-135a2 gene by binding to an androgen response element in the promoter region. Our findings identify miR-135a as a novel effector in androgens/AR signaling. Using xenograft experiments in chick embryos and adult male mice, we showed that miR-135a overexpression decreases in vivo invasion abilities of prostate PC-3 cells. Through in vitro wound-healing migration and invasion assays, we demonstrated that this effect is mediated through downregulating ROCK1 and ROCK2 expression, two genes that we characterized as miR-135a direct target genes. In human surgical samples from prostatectomy, we observed that miR-135a expression was lower in tumoral compared with paired adjacent normal tissues, mainly in tumors classified with a high Gleason score (⩾8). Moreover, miR-135a expression is lower in invasive tumors, showing extraprostatic extension, as compared with intraprostatic localized tumors. In tumor relative to normal glands, we also showed a more frequently higher ROCK1 protein expression determined using a semi-quantitative immunohistochemistry analysis. Therefore, in tumor cells, the lower miR-135a expression could lead to a higher ROCK1 protein expression, which could explain their invasion abilities. The highlighted relationship between miR-135a expression level and the degree of disease aggressiveness suggests that miR-135a may be considered as a prognostic marker in human PCa.

Retinoic acid and mouse skin morphogenesis. I. Expression pattern of retinoic acid receptor genes during hair vibrissa follicle, plantar, and nasal gland development.

The spatial and temporal expression of the nuclear retinoic acid receptors alpha, beta, and gamma (RAR-alpha, beta, and gamma) was compared by in situ hybridization during hair vibrissa follicle and nasal and plantar eccrine gland morphogenesis in mouse embryo. The RAR-alpha and RAR-gamma transcripts are abundant in the dermal papilla cells of the hair vibrissa when these cells elicit epidermal hair placode (12.5-d embryos) and hair follicle (13.5-d embryos) formation. Both these transcripts are also abundant in the dermal cells of the plantar foot pad at the initiation stage (17.5-d embryos) of glandular morphogenesis. In epidermal cells, the distribution of RAR-gamma transcripts increases in parallel with hair vibrissa follicle and sweat gland differentiation, and thus may be part of the epidermal response to the dermal instructions. The RAR-beta signal is barely above control level during both hair vibrissa and plantar gland morphogenesis. By contrast, during nasal gland formation (12.5- to 15.5-d embryos), the RAR-beta signal reaches a high level in mesenchymal cells, whereas the RAR-alpha-transcripts are present in both epithelial and mesenchymal cells. These results suggest a role for RAR-alpha and RAR-gamma in the epidermal-dermal interactions that lead to hair follicle and plantar gland morphogenesis, whereas the nasal gland development implies RAR-alpha and RAR-beta gene expression. This should be correlated with the expression of the RAR-beta gene that was previously shown to be linked to the RA-induced glandular metaplasia of hair vibrissa follicles.

Differential expression pattern of the three Fringe genes is associated with epidermal differentiation.

Epidermal differentiation, as keratinocytes go through different layers to the skin surface, may imply a differential activation of Notch transmembrane proteins. In mouse, as recently shown in Drosophila, Notch activation by its ligands may be modulated by Fringe secreted proteins. Therefore, we cloned the mouse homolog of Radical-fng, synthesized riboprobes for Lunatic-fng, Manic-fng, and Radical-fng, and examined their expression during epidermal differentiation. Expression of all three genes is differentially activated during embryonic epidermal stratification. Manic-fng and Lunatic-fng are expressed in the basal layer, whereas Lunatic-fng is expressed in the granular layer and Radical-fng is restricted to the most differentiated nucleated layer. This expression decreases by a few days postnatally and can be reactivated by retinoic acid treatment, which triggers a new distribution of Fringe transcripts and a thickening of the granular layer. Therefore, Manic, Lunatic, and Radical Fringe by modulating the Notch pathway may play a key role in defining the different steps of keratinocyte differentiation.

A 60-kilodalton protein in rat hepatoma cells overexpressing insulin receptor was tyrosine phosphorylated and associated with Syp, phophatidylinositol 3-kinase, and Grb2 in an insulin-dependent manner.

Tyrosine phosphorylation of cellular proteins is an early and key step after activation of the insulin receptor kinase (IRK). The study of the properties of these proteins should contribute to our understanding of insulin action. In rat hepatoma cells overexpressing human insulin receptors (HTC-IR), insulin treatment resulted in rapid tyrosine phosphorylation of proteins of 180, 94, 68, and 60 kDa. When lysates from insulin-treated cells were immunoprecipitated with anti-Syp antibody, subsequent immunoblotting identified p65 and p68, which reacted with anti-Syp, and p6O and p68, which reacted with antiphosphotyrosine antibody. Thus, insulin treatment yielded tyrosine phosphorylation of both Syp and a Syp-associated p6O molecule. When lysates from insulin-treated cells were adsorbed with a glutathione S-transferase (GST)-Syp-Src homology-2 (SH2) fusion protein, tyrosine- phosphorylated p6O was sequestered. After subjecting lysates to SDS-PAGE, the GST-SypSH2 fusion protein was found to bind to p18O, p94, and p6O. Thus, Syp associates directly with a 60-kDa IRK substrate via its SH2 domains. Syp-associated p6O differed from the 60- to 62-kDa proteins, associating with ras guanosine triphosphatase-activating protein, which also underwent modest tyrosine phosphorylation in response to insulin. Preadsorption of cell lystates with antibody against the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase substantially reduced the amount of p60 subsequently immunoprecipitated by anti-Syp. Thus, p60 associates with both Syp and p85. The amount of tyrosine-phosphorylated p60 exceeded that of p180 in anti-Syp immunoprecipitates, whereas their proportion was comparable in anti-p85 immunoprecipitates. Grb2 was also observed in the anti-Syp immunoprecipitates. When lysates from insulin-treated cells were adsorbed with GST-p85SH2 domains or GST-Grb2, the subsequent eluates contained tyrosine-phosphorylated p60, as determined by immunoblotting with antiphosphotyrosine. Membrane binding assays using GST fusion proteins showed that these associations were direct. Studies in rat liver, muscle, and adipose tissue identified insulin-dependent association of Syp, Grb2, and p85 with tyrosine-phosphorylated p60 in adipose tissue only. We conclude that insulin treatment of HTC-IR cells and rat adipose tissue results in the tyrosine phosphorylation of p60, which might participate in the recruitment of downstream effectors involved in insulin signal transduction.

Bombesin specifically induces intracellular calcium mobilization via gastrin-releasing peptide receptors in human prostate cancer cells.

Bombesin and gastrin-releasing peptide (GRP) are potent neuropeptides expressed by prostate cancer neuroendocrine cells and are related to the progression of this malignancy. This study characterizes bombesin receptors in human prostate cancer cell lines (PC-3, DU-145, LNCaP) and assesses the in vitro effect of bombesin on signal transduction and cell proliferation. [125I]Tyr4-bombesin binding assays (37 degrees C) and Scatchard analyses revealed the presence of a single class of high-affinity receptors with similar Kd values (1.5, 1.1 and 3.6 x 10(-10) M in PC-3, DU-145 and LNCaP cells respectively) but with significant differences in the number of binding sites per cell (47.6, 1.5 and 0.1 x 10(3) in PC-3, DU-145 and LNCaP cells respectively). Molecular characterization of the binding sites performed in PC-3 cells by cross-linking experiments and SDS/PAGE revealed a single radioactive band of 85 kDa. To determine which of the three known bombesin receptor subtypes (GRP receptor (GRP-R), neuromedin B receptor, bombesin receptor subtype-3) were expressed in the cell lines, reverse transcription/PCR analysis of cellular RNA followed by hybridization with receptor-specific cDNA was performed. This revealed the presence of GRP-R transcript in all cell lines, while neither of the other two receptor transcripts were expressed. When intracellular calcium mobilization was measured by Fura-2/AM cell labeling and spectrofluorometric monitoring, bombesin (100 nM) induced rapid calcium mobilization in both PC-3 (> 200% of baseline) and DU-145 (> 100% of baseline) cells, but not in LNCaP cells. However, as measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and [3H]thymidine incorporation, no growth modulation was observed with bombesin or bombesin receptor antagonist at various concentrations (0-500 nM). Our data indicate that bombesin is a potent inducer of signal transduction via GRP-R receptors in androgen-insensitive PC-3 and DU-145 prostate cancer cells. This suggests that the bombesin/GRP family of neuropeptides may play a regulatory role in the biology of androgen-independent prostate cancer.