Cancer immunotherapy with immune checkpoint inhibitors (ICIs): potential, mechanisms of resistance, and strategies for reinvigorating T cell responsiveness when resistance is acquired.

Cancer is still the leading cause of death globally. The approval of the therapeutic use of monoclonal antibodies against immune checkpoint molecules, notably those that target the proteins PD-1 and PD-L1, has changed the landscape of cancer treatment. In particular, first-line PD-1/PD-L1 inhibitor drugs are increasingly common for the treatment of metastatic cancer, significantly prolonging patient survival. Despite the benefits brought by immune checkpoint inhibitors (ICIs)-based therapy, the majority of patients had their diseases worsen following a promising initial response. To increase the effectiveness of ICIs and advance our understanding of the mechanisms causing cancer resistance, it is crucial to find new, effective, and tolerable combination treatments. In this article, we addressed the potential of ICIs for the treatment of solid tumors and offer some insight into the molecular pathways behind therapeutic resistance to ICIs. We also discuss cutting-edge therapeutic methods for reactivating T-cell responsiveness after resistance has been established.

Exome sequencing of glioblastoma-derived cancer stem cells reveals rare clinically relevant frameshift deletion in MLLT1 gene.

BACKGROUND: Glioblastoma multiforme (GBM) is a heterogeneous CNS neoplasm which causes significant morbidity and mortality. One reason for the poor prognostic outcome of GBM is attributed to the presence of cancer stem cells (CSC) which confer resistance against standard chemo- and radiotherapeutics modalities. Two types of GBM-associated CSC were isolated from the same patient: tumor core- (c-CSC) and peritumor tissue-derived cancer stem cells (p-CSC). Our experiments are focused on glioblastoma-IDH-wild type, and no disease-defining alterations were present in histone, BRAF or other genes. METHODS: In the present study, potential differences in genetic variants between c-CSC versus p-CSC derived from four GBM patients were investigated with the aims of (1) comparing the exome sequences between all the c-CSC or p-CSC to identify the common variants; (2) identifying the variants affecting the function of genes known to be involved in cancer origin and development. RESULTS: By comparative analyses, we identified common gene single nucleotide variants (SNV) in all GBM c-CSC and p-CSC, a potentially deleterious variant was a frameshift deletion at Gln461fs in the MLLT1 gene, that was encountered only in p-CSC samples with different allelic frequency. CONCLUSIONS: We discovered a potentially harmful frameshift deletion at Gln461fs in the MLLT1 gene. Further investigation is required to confirm the presence of the identified mutations in patient tissue samples, as well as the significance of the frameshift mutation in the MLLT1 gene on GBM biology and response to therapy based on genomic functional experiments.

p53 signaling in cancer progression and therapy.

The p53 protein is a transcription factor known as the "guardian of the genome" because of its critical function in preserving genomic integrity. The TP53 gene is mutated in approximately half of all human malignancies, including those of the breast, colon, lung, liver, prostate, bladder, and skin. When DNA damage occurs, the TP53 gene on human chromosome 17 stops the cell cycle. If p53 protein is mutated, the cell cycle is unrestricted and the damaged DNA is replicated, resulting in uncontrolled cell proliferation and cancer tumours. Tumor-associated p53 mutations are usually associated with phenotypes distinct from those caused by the loss of the tumor-suppressing function exerted by wild-type p53protein. Many of these mutant p53 proteins have oncogenic characteristics, and therefore modulate the ability of cancer cells to proliferate, escape apoptosis, invade and metastasize. Because p53 deficiency is so common in human cancer, this protein is an excellent option for cancer treatment. In this review, we will discuss some of the molecular pathways by which mutant p53 proteins might perform their oncogenic activities, as well as prospective treatment methods based on restoring tumor suppressive p53 functions.

Pandemic COVID-19 caused by SARS-CoV-2: genetic structure, vaccination, and therapeutic approaches.

We give a summary of SARS-genetic CoV-2's structure and evolution, as well as current attempts to develop efficient vaccine and treatment methods for SARS-CoV-2 infection, in this article. Most therapeutic strategies are based on repurposing of existing therapeutic agents used against various virus infections and focused mainly on inhibition of the virus replication cycle, enhancement of innate immunity, and alleviation of CRS caused by COVID-19. Currently, more than 100 clinical trials on COVID-19 aim to provide robust evidence on the efficacy of the currently available anti-SARS-CoV-2 antiviral substances, such as the nucleotide analogue remdesivir, the antimalarial drug chloroquine, and drugs directed against docking of SARS-CoV-2 to the membrane-associated angiotensin-converting enzyme 2 (ACE2) such as transmembrane protease serine 2 (TMPRSS2). The current vaccination campaign is ongoing worldwide using different types of vaccines such as Pfizer-BioNTech and Moderna, Johnson & Johnson, Oxford-AstraZeneca, Novavax, and others with efficacy ranging from 72-95%. In March 2021 Germany limited the use of the Oxford-AstraZeneca COVID-19 vaccine to people 60 years of age and older due to concerns that it may be causing blood clots. Further study and more data are needed to confirm the safety of different available vaccines.

Aspirin inhibits proliferation and promotes differentiation of neuroblastoma cells via p21Waf1 protein up-regulation and Rb1 pathway modulation.

Several clinical and experimental studies have demonstrated that regular use of aspirin (acetylsalicylic acid, ASA) correlates with a reduced risk of cancer and that the drug exerts direct anti-tumour effects. We have previously reported that ASA inhibits proliferation of human glioblastoma multiforme-derived cancer stem cells. In the present study, we analysed the effects of ASA on nervous system-derived cancer cells, using the SK-N-SH (N) human neuroblastoma cell line as an experimental model. ASA treatment of SK-N-SH (N) dramatically reduced cell proliferation and motility, and induced neuronal-like differentiation, indicated by the appearance of the neuronal differentiation marker tyrosine hydroxylase (TH) after 5 days. ASA did not affect cell viability, but caused a time-dependent accumulation of cells in the G0 /G1 phase of the cell cycle, with a concomitant decrease in the percentage of cells in the G2 phase. These effects appear to be mediated by a COX-independent mechanism involving an increase in p21Waf1 and underphosphorylated retinoblastoma (hypo-pRb1) protein levels. These findings may support a potential role of ASA as adjunctive therapeutic agent in the clinical management of neuroblastoma.

Aspirin inhibits cancer stem cells properties and growth of glioblastoma multiforme through Rb1 pathway modulation.

Several clinical studies indicated that the daily use of aspirin or acetylsalicylic acid reduces the cancer risk via cyclooxygenases (Cox-1 and Cox-2) inhibition. In addition, aspirin-induced Cox-dependent and -independent antitumor effects have also been described. Here we report, for the first time, that aspirin treatment of human glioblastoma cancer (GBM) stem cells, a small population responsible for tumor progression and recurrence, is associated with reduced cell proliferation and motility. Aspirin did not interfere with cell viability but induced cell-cycle arrest. Exogenous prostaglandin E2 significantly increased cell proliferation but did not abrogate the aspirin-mediated growth inhibition, suggesting a Cox-independent mechanism. These effects appear to be mediated by the increase of p21 waf1 and p27 Kip1 , associated with a reduction of Cyclin D1 and Rb1 protein phosphorylation, and involve the downregulation of key molecules responsible for tumor development, that is, Notch1, Sox2, Stat3, and Survivin. Our results support a possible role of aspirin as adjunctive therapy in the clinical management of GBM patients.

The human microglial HMC3 cell line: where do we stand? A systematic literature review.

Microglia, unique myeloid cells residing in the brain parenchyma, represent the first line of immune defense within the central nervous system. In addition to their immune functions, microglial cells play an important role in other cerebral processes, including the regulation of synaptic architecture and neurogenesis. Chronic microglial activation is regarded as detrimental, and it is considered a pathogenic mechanism common to several neurological disorders. Microglial activation and function have been extensively studied in rodent experimental models, whereas the characterization of human cells has been limited due to the restricted availability of primary sources of human microglia. To overcome this problem, human immortalized microglial cell lines have been developed. The human microglial clone 3 cell line, HMC3, was established in 1995, through SV40-dependent immortalization of human embryonic microglial cells. It has been recently authenticated by the American Type Culture Collection (ATCC®) and distributed under the name of HMC3 (ATCC®CRL-3304). The HMC3 cells have been used in six research studies, two of which also indicated by ATCC® as reference articles. However, a more accurate literature revision suggests that clone 3 was initially distributed under the name of CHME3. In this regard, several studies have been published, thus contributing to a more extensive characterization of this cell line. Remarkably, the same cell line has been used in different laboratories with other denominations, i.e., CHME-5 cells and C13-NJ cells. In view of the fact that "being now authenticated by ATCC®" may imply a wider distribution of the cells, we aimed at reviewing data obtained with the human microglia cell line clone 3, making the readers aware of this complicated nomenclature. In addition, we also included original data, generated in our laboratory with the HMC3 (ATCC®CRL-3304) cells, providing information on the current state of the culture together with supplementary details on the culturing procedures to obtain and maintain viable cells.

Generation of gene edited hiPSC from familial Alzheimer's disease patient carrying N141I missense mutation in presenilin 2.

Alzheimer's disease (AD) is the major cause of dementia worldwide. Early-onset familial AD accounts for about 0.5% of all AD and is caused by single major gene mutations and autosomal dominant inheritance. An N141I missense mutation is associated with a significant increase in basal cell death and apoptosis. In this work we generated hiPSC from skin fibroblasts obtained from an AD patient carrying a N141I missense mutation in PSEN2. The generated iPSC colonies grew and were characterized by pluripotency marker staining; the N141I missense mutation was corrected using genome editing technology.

Current progress in chimeric antigen receptor T cell therapy for glioblastoma multiforme.

Glioblastoma multiforme (GBM) is one of the deadliest brain tumors with an unfavorable prognosis and overall survival of approximately 20 months following diagnosis. The current treatment for GBM includes surgical resections and chemo- and radiotherapeutic modalities, which are not effective. CAR-T immunotherapy has been proven effective for CD19-positive blood malignancies, and the application of CAR-T cell therapy for solid tumors including GBM offers great hope for this aggressive tumor which has a limited response to current treatments. CAR-T technology depends on the use of patient-specific T cells genetically engineered to express specific tumor-associated antigens (TAAs). Interaction of CAR-T cells with tumor cells triggers the destruction/elimination of these cells by the induction of cytotoxicity and the release of different cytokines. Despite the great promise of CAR-T cell-based therapy several challenges exist. These include the heterogeneity of GBM cancer cells, aberrant various signaling pathways involved in tumor progression, antigen escape, the hostile inhibitory GBM microenvironment, T cell dysfunction, blood-brain barrier, and defective antigen presentation. All need to be addressed before full application at the clinical level can begin. Herein we provide a focused review of the rationale for the use of different types of CAR-T cells (including FcγRs), the different GBM-associated antigens, the challenges still facing CAR-T-based therapy, and means to overcome such challenges. Finally, we enumerate currently completed and ongoing clinical trials, highlighting the different ways such trials are designed to overcome specific problems. Exploitation of the full potential of CAR-T cell therapy for GBM depends on their solution.

Erythropoietin inhibits basal and stimulated corticotropin-releasing hormone release from the rat hypothalamus via a nontranscriptional mechanism.

Brain hypoxia-ischemia induces a local increase in the levels of erythropoietin (EPO) and vascular endothelial growth factor (VEGF); this condition is also associated with acute activation of the hypothalamo-pituitary-adrenal (HPA) axis, suggesting that increased levels of EPO and VEGF in the hypothalamus may play a role in the control of HPA function. Thus, in this study we used rat hypothalamic explants to investigate whether EPO and VEGF can directly modulate CRH release; the latter was assessed by RIA measurement of the peptide in the incubation medium and hypothalamic tissue. EPO and VEGF effects were studied in short-term (1-3 h) experiments under basal conditions or after stimulation with 56 mM KCl or 10 microM veratridine. We observed that EPO (1-10 nm) significantly reduced CRH release and, in parallel, increased intrahypothalamic CRH content. VEGF tended to reduce CRH release without reaching statistical significance. Moreover, EPO, but not VEGF, inhibited KCl- and veratridine-stimulated CRH release and counteracted the parallel decrease in intrahypothalamic CRH induced by the two secretagogues. EPO effects were not mediated by modification of CRH gene expression, either in the absence or the presence of KCl or veratridine; in this paradigm, KCl and veratridine per se did not modify CRH gene expression. Our findings suggest that EPO contributes to the regulation of the HPA axis activation; in pathological conditions such as brain ischemia, this growth factor may control the HPA axis function, preventing possible detrimental effects of HPA overactivation.

Evidence that corticotropin-releasing hormone inhibits cell growth of human breast cancer cells via the activation of CRH-R1 receptor subtype.

It has been previously shown that corticotropin-releasing hormone (CRH) exerts antiproliferative activity on an estrogen-dependent tumor cell line, i.e. human endometrial adenocarcinoma Ishikawa (IK) cells. Here we have investigated the effects of CRH on another estrogen-dependent tumor cell line, human breast cancer MCF7 cells. In this paradigm, CRH given at a fixed concentration of 100 nM significantly inhibited cell growth induced by 100 nM estradiol (E2) after 48 and 72 h of incubation. This effect was not associated with the induction of apoptosis. CRH inhibition of cell proliferation was counteracted in a concentration-dependent manner by the non-selective CRH receptor antagonist, astressin, as well as by a CRH-R1 selective receptor antagonist, antalarmin. RNase protection assays carried out on MCF7 under basal conditions showed that these cells express in a constitutive manner the CRH-R1 receptor subtype. We have also investigated the putative source of CRH acting on breast cancer cells; we found that MCF7 cells express CRH mRNA under basal conditions and secrete sizable amounts of immunoreactive CRH, which leads to postulate the existence of paracrine-autocrine inhibitory mechanism operated by CRH in breast cancer cells.

Hydroxyurea induces vasopressin release and cytokine gene expression in the rat hypothalamus.

We previously showed that the cytostatic drug hydroxyurea (HU) activates the hypothalamo-pituitary-adrenal (HPA) axis in intact rats, whereas it is lethal in rats with impaired HPA function. In these animals, HU toxicity is mediated by increased circulating levels of proinflammatory cytokines, whose secretion cannot be counteracted by glucocorticoids, suggesting that HPA activation blunts HU toxicity. Here we investigated the mechanisms through which HU activates the HPA axis, looking at the direct effects of the drug on the isolated hypothalamus. We found that HU significantly increases the release of arginine vasopressin but not that of corticotrophin-releasing hormone in short-term incubation experiments. The levels of arginine vasopressin are also increased in the hypothalamus and systemic circulation 2 h after the in vivo administration of the drug. Furthermore, HU increased significantly the expression of interleukin-6 and, to a lesser extent, interleukin-1beta in the hypothalamus. Interestingly, experiments with HU on primary cultures of rat microglia and astrocytes suggested that the increase in cytokine gene expression observed in hypothalamic explants is not accounted for by glial cells. Since both vasopressin and cytokines can activate the HPA axis, our present findings provide a reasonable explanation of the HPA activation elicited by HU in vivo in the rat.

Lamotrigine inhibits basal and Na+-stimulated, but not Ca2+-stimulated, release of corticotropin-releasing hormone from the rat hypothalamus.

RATIONALE: Corticotropin-releasing hormone (CRH) is a peptide neurotransmitter involved in the pathogenesis of anxiety and depressive disorders; CRH receptor antagonists are currently developed as anxiolytic and antidepressive agents, and several antidepressants negatively modulate CRH in the central nervous system. OBJECTIVES AND METHODS: Originally marketed as an antiepileptic drug, lamotrigine (LTG) was subsequently shown to be effective as a mood stabilizer and an antidepressant. In this study, we used acute rat hypothalamic explants to investigate the effects of LTG on the gene expression and secretion of CRH from the hypothalamus in short-term incubation experiments. RESULTS: We found that LTG reduces basal CRH release in a time- and concentration-dependent manner. LTG also inhibits veratridine-stimulated, but not K+-stimulated, CRH release in 1-h experiments. Moreover, LTG tended to reduce CRH mRNA expression in 1-h experiments, regardless of whether the drug was given alone or in combination with veratridine or high K+ concentrations; such reduction achieved statistical significance after 3 h of incubation. CONCLUSION: In 1-h experiments, LTG reduces CRH release from CRH-containing neurons in the rat hypothalamus by interfering with Na+-driven secretion mechanisms. After 3-h incubations, the reduction in CRH release is also accounted for by LTG-induced decrease in CRH gene expression.

Fragment 31-35 of beta-amyloid peptide induces neurodegeneration in rat cerebellar granule cells via bax gene expression and caspase-3 activation. A crucial role for the redox state of methionine-35 residue.

The amyloid beta-peptide (AbetaP) is the major protein component of brain senile plaques in Alzheimer's disease. The redox state of methionine-35 residue plays a critical role in peptide neurotoxic actions. We used the fragment 31-35 of AbetaP [AbetaP(31-35)], containing a single methionine-35 residue (Met-35), to investigate the relationship between the oxidative state of Met-35 and neurotoxic and pro-apoptotic actions induced by the peptide; in rat cerebellar granule cells (CGC), we compared the effects of AbetaP(31-35), in which the Met-35 is present in the reduced state, with those of a modified peptide with oxidized Met-35 [AbetaP(31-35)Met-35(OX)](,) as well as an AbetaP-derivative with Met-35 substituted by norleucine [AbetaP(31-35)Nle-35]. AbetaP(31-35) induced a time-dependent decrease in cell viability. AbetaP(31-35)Met-35(OX) was significantly less potent, but still induced a significant decrease in cell viability compared to control. No toxic effects were observed after treatment with AbetaP(31-35)Nle-35. AbetaP(31-35) induced a 2-fold increase in bax mRNA levels after 4h, whereas AbetaP(31-35)Met-35(OX) raised bax mRNA levels by 41% and AbetaP(31-35)Nle-35 had no effect. Finally, AbetaP(31-35) caused a 43% increase in caspase-3 activity after 24h; AbetaP(31-35)Met-35(OX) caused only a 18% increase, and AbetaP(31-35)Nle-35 had no effect. These findings suggest that AbetaP(31-35)-induced neurodegeneration in CGC is mediated by a selective early increase in bax mRNA levels followed by delayed caspase-3 activation; the redox state of the single Met-35 residue is crucial in the occurrence and extent of the above phenomena.

Corticotropin-releasing hormone receptor-1 in human endometrial cancer.

We have previously shown that corticotrophin-releasing hormone (CRH) inhibits the proliferation of Ishikawa (IK) human endometrial carcinoma cell line through the activation of CRH-R1 receptors. Here, we have further investigated the role of CRH and its type-1 receptor in the control of IK cell function, and we carried out a pilot study in tumor tissues obtained from 19 patients with endometrial cancer, looking at CRH-R1 gene expression. In the IK study, CRH counteracted the increase in cell proliferation caused by estradiol; type-1 receptors mediating this effect belong to the alpha subtype. In the study on human tumors, CRH-R1 was expressed in 4 out of 19 (21%) surgical specimens obtained from untreated patients with a diagnosis of primary endometrial cancer. Two out of 4 cases (50%) expressing CRH-R1 mRNA had extrauterine spreading of the disease, whereas cases not expressing CRH-R1 mRNA were all FIGO stage I, 2 (p=0.015).

Metallic but not ceramic wear particles increase prostaglandin E2 release and interleukin-1beta gene expression in human blood monocytes in vitro.

In this study the potential of clinically relevant alumina ceramic and metal wear particles to induce an in vitro inflammatory response was assessed in human monocytes and lymphocytes isolated from healthy donors by measuring prostaglandin E2 (PGE2) levels and mRNA expression of various pro-inflammatory cytokines. Bacterial lipopolysaccharide (LPS) was used as positive control. LPS significantly increased PGE2 levels in the incubation medium of monocyte cultures after 24 h. Alumina had no effect on PGE2 production, whereas metals induced a concentration-dependent increase in PGE2 release, that was statistically significant at the dose of 0.1 mg/ml. In lymphocytes, LPS elicited a weak but significant increase in PGE2 release, whereas both alumina and metals did not modify PGE2 amounts at any of the concentrations tested. The gene expression of a number of pro- and anti-inflammatory cytokines was assessed in monocytes and lymphocytes exposed to LPS, 0.1 mg/ml alumina or 0.1 mg/ml metals for 24 h. In monocytes, LPS caused a 2-fold increase in interleukin-1beta (IL-1beta) mRNA levels. The exposure of monocytes to metals resulted in a selective increase in IL-1beta mRNA accumulation (+48% compared to control). By contrast, alumina did not modify IL-1beta mRNA levels. None of the test substances elicited any response on purified lymphocyte population. These findings suggest that PGE2 production and IL-1 mRNA expression are a reliable marker to study the pro-inflammatory effects of wear debris in vitro. The lower activity of alumina compared to metals suggests that the former should be preferred in implants for its favorable biological and mechanical behavior.

Interleukin-18 displays effects opposite to those of interleukin-1 in the regulation of neuroendocrine stress axis.

The effects of interleukin-18 (IL-18), a putative member of the IL-1 family, were investigated on basal and stimulated release of corticotropin-releasing hormone (CRH) and prostanoids from rat hypothalamic explants and glial cells in vitro. We found that IL-18 decreases basal and KCl-stimulated CRH release from the hypothalamus. IL-18 also reduced CRH gene expression after 1- and 3-h incubation. The cytokine did not modify basal PGE2 production by hypothalamic explants but abolished production stimulated by IL-1beta. Similar effects were also observed on cultured glial cells. The present findings show that IL-18 possesses a profile of in vitro neuroendocrine activities opposing to, and even antagonizing, those of IL-1beta.

Mirtazapine acutely inhibits basal and K(+)-stimulated release of corticotropin-releasing hormone from the rat hypothalamus via a non-genomic mechanism.

RATIONALE: Originally described as a pivotal mediator of acute neuroendocrine responses to stress, corticotropin-releasing hormone (CRH) is currently envisioned as a peptide neurotransmitter involved in the pathogenesis of anxiety and depressive disorders; it has been postulated that antidepressant drugs are clinically effective insofar as they are able to reduce central CRH production and release. OBJECTIVES AND METHODS: In this study we used a well validated in vitro model, i.e. acute rat hypothalamic explants, to investigate the effects of the antidepressant mirtazapine on the production and release of CRH from the hypothalamus in short-term experiments. CRH release was assessed through the measurement of CRH immunoreactivity in the incubation medium. RESULTS: We found that mirtazapine reduces in a concentration-dependent manner both basal and K(+)-stimulated CRH release in 30-min and 60-min experiments. Mirtazapine had no effect on CRH mRNA expression in 1-h and 3-h experiments; the intra-hypothalamic levels of peptide were not reduced, and even tended to increase, with respect to controls. CONCLUSION: Mirtazapine reduces CRH release from CRH-containing neurons in the rat hypothalamus through a mechanism independent from the modulation of CRH gene expression and peptide production.

The activation of type 1 corticotropin releasing factor receptor (CRF-R1) inhibits proliferation and promotes differentiation of neuroblastoma cells in vitro via p27(Kip1) protein up-regulation and c-Myc mRNA down-regulation.

Our group has previously shown that corticotropin releasing factor (CRF) inhibits proliferation of human endocrine-related cancer cell lines via the activation of CRF type-1 receptors (CRF-R1). Tumors originating from the nervous system also express CRF receptors but their role on neoplastic cell proliferation was poorly investigated. Here we investigated the effect of CRF receptor stimulation on nervous system-derived cancer cells, using the SK-N-SH (N) human neuroblastoma cell line as an experimental model. We found that SK-N-SH (N) cells express functionally active CRF-R1, whose activation by CRF and the cognate peptide urocortin (UCN) is associated to reduced cell proliferation and motility, as well as neuronal-like differentiation. UCN did not interfere with cell viability and cell-cycle arrest. Those effects seem to be mediated by a mechanism involving the activation of cAMP/PKA/CREB pathway and the subsequent downstream increase in p27(Kip1) and underphosphorylated retinoblastoma protein levels, as well as reduced c-Myc mRNA accumulation.

CRH inhibits cell growth of human endometrial adenocarcinoma cells via CRH-receptor 1-mediated activation of cAMP-PKA pathway.

CRH produced by human endometrial cells exerts decidualizing activity via an autocrine mechanism mediated via CRH-R1 receptors. We postulated that such activity exerted by CRH on normal endometrial cells might translate into an antiproliferative action on endometrial-derived malignancies, provided that neoplastic cells maintain the expression of CRH receptors. In this light, here we investigated the possible antiproliferative effects of CRH in an adenocarcinoma cell line derived from human endometrium. CRH induces time- and concentration-dependent inhibition of Ishikawa cell growth, the maximal effect (50% inhibition) being achieved after 3 d of treatment with 10(-7) M CRH. A decrease in telomerase activity, which paralleled tumor growth inhibition, was also observed in CRH-treated samples. The antiproliferative effect was confirmed by colony-formation assay for long-term survival. This effect was counteracted in a concentration-dependent manner by both alpha-helical CRH and astressin; the former also showed intrinsic inhibitory activity. These findings suggested the involvement of CRH-R1 receptor subtype; this hypothesis was confirmed by RNase protection analysis showing the expression of human CRH-R1 mRNA. Experiments with the PKA inhibitor 14-22 amide and forskolin, as well as the measurement of intracellular cAMP, suggested the downstream involvement of cAMP-PKA pathway in CRH-induced inhibition of Ishikawa cell growth.