Clinical and molecular delineation of the 17q21.31 microdeletion syndrome.

BACKGROUND: The chromosome 17q21.31 microdeletion syndrome is a novel genomic disorder that has originally been identified using high resolution genome analyses in patients with unexplained mental retardation. AIM: We report the molecular and/or clinical characterisation of 22 individuals with the 17q21.31 microdeletion syndrome. RESULTS: We estimate the prevalence of the syndrome to be 1 in 16,000 and show that it is highly underdiagnosed. Extensive clinical examination reveals that developmental delay, hypotonia, facial dysmorphisms including a long face, a tubular or pear-shaped nose and a bulbous nasal tip, and a friendly/amiable behaviour are the most characteristic features. Other clinically important features include epilepsy, heart defects and kidney/urologic anomalies. Using high resolution oligonucleotide arrays we narrow the 17q21.31 critical region to a 424 kb genomic segment (chr17: 41046729-41470954, hg17) encompassing at least six genes, among which is the gene encoding microtubule associated protein tau (MAPT). Mutation screening of MAPT in 122 individuals with a phenotype suggestive of 17q21.31 deletion carriers, but who do not carry the recurrent deletion, failed to identify any disease associated variants. In five deletion carriers we identify a <500 bp rearrangement hotspot at the proximal breakpoint contained within an L2 LINE motif and show that in every case examined the parent originating the deletion carries a common 900 kb 17q21.31 inversion polymorphism, indicating that this inversion is a necessary factor for deletion to occur (p<10(-5)). CONCLUSION: Our data establish the 17q21.31 microdeletion syndrome as a clinically and molecularly well recognisable genomic disorder.

US military medical ethics in the War on Terror.

Military medical ethics has been challenged by the post-11 September 2001 'War on Terror'. Two recurrent questions are whether military physicians are officers first or physicians first, and whether military physicians need a separate code of ethics. In this article, we focus on how the War on Terror has affected the way we have addressed these questions since 2001. Two examples frame this discussion: the use of military physicians to force-feed hunger strikers held in Guantanamo Bay prison camp, and the uncertain fate of the Department of Defense's report on 'Ethical Guidelines and Practices for US Military Medical Professionals'.

Prevalence of hemangiosarcoma in anemic dogs with a splenic mass and hemoperitoneum requiring a transfusion: 71 cases (2003-2005).

OBJECTIVE: To determine prevalence of splenic hemangiosarcoma in anemic dogs with a splenic mass and hemoperitoneum requiring a transfusion and to identify factors that could differentiate between dogs with hemangiosarcoma and dogs with other splenic masses at the time of hospital admission. DESIGN: Retrospective case series. ANIMALS: 71 dogs. PROCEDURES: Medical records, blood bank logs, and histologic reports of dogs with a splenic mass and hemoperitoneum that required a transfusion between 2003 and 2005 were reviewed. Dogs that received a transfusion of packed RBCs, were splenectomized, and had a definitive histologic diagnosis were included. RESULTS: Signalment of dogs was similar to that in other reports. Malignant splenic neoplasia was identified in 54 of 71 (76.1%) dogs, whereas 17 of 71 (23.9%) dogs had a benign splenic lesion. Of 54 dogs with malignant splenic neoplasia, 50 (92.6% [70.4% of all dogs]) had splenic hemangiosarcoma. In addition, dogs with splenic hemangiosarcoma had significantly lower total solids (TS) concentrations and platelet counts at admission. Finally, hemoperitoneum was strongly associated with a diagnosis of splenic hemangiosarcoma. CONCLUSIONS AND CLINICAL RELEVANCE: In this clinical population of dogs, prevalence of hemangiosarcoma was higher than in other studies. Dogs with hemangiosarcoma in this study had significantly lower TS concentrations and platelet counts at the time of admission, compared with values for dogs with other splenic masses. No other markers were useful in differentiating dogs with hemangiosarcoma. It is important to discuss the prevalence of and poor prognosis associated with hemangiosarcoma with owners when they are contemplating whether to proceed with treatment.

Comparative efficacy of enrofloxacin to that of tulathromycin for the control of bovine respiratory disease and prevalence of antimicrobial resistance in Mannheimia haemolytica in calves at high risk of developing bovine respiratory disease.

Bovine respiratory disease (BRD) is the most common cause of morbidity and mortality in North American beef cattle. Mannheimia haemolytica is the bacterial pathogen most often isolated from cattle with BRD, and the prevalence of antimicrobial resistance (AMR) in this organism has increased in recent years. Antimicrobials are commonly used to prevent BRD in cattle at high risk of developing BRD; however, recent work would suggest that this practice might be one factor contributing to the increased prevalence of AMR in M. haemolytica. We hypothesized that the administration of the short-acting fluoroquinolone, enrofloxacin, would be just as effective as the long-acting triamilide, tulathromycin, in preventing BRD but would be less likely to select for AMR M. haemolytica in stocker calves at high risk of developing BRD. Three hundred forty-one stocker calves were enrolled in the study with 172 calves in 4 pens being randomly assigned to treatment with enrofloxacin and 169 calves in 4 pens randomly assigned to treatment with tulathromycin. Calves within each treatment group were allocated to one of 4 replicate pens based on the week of enrollment. Of calves receiving enrofloxacin, 33.7% required treatment for BRD at least once within 45 d after arrival, compared with 18.3% of calves receiving tulathromycin (P = 0.040). The percentages of calves that required more than one treatment for BRD within 45 d after arrival did not differ statistically for those receiving enrofloxacin compared with those receiving tulathromycin (10.5% and 4.7%, respectively; P = 0.107) Likewise, the percentages of calves that died during the 45-d follow-up period did not differ for those receiving enrofloxacin compared with those receiving tulathromycin (12.2% and 10.1%, respectively; P = 0.592). Mannheimia haemolytica was cultured from 11% of calves sampled at arrival and from 50% of calves sampled at revaccination 14 to 17 d later. There was a significanst effect of sampling time on the proportion of calves carrying multidrug-resistant (MDR) isolates, with calves having a higher prevalence of MDR isolates at revaccination than arrival (100% vs. 13%; P < 0.001). Future research evaluating the impact of MDR on response to antimicrobial therapy is necessary.

Learning from UJAMBO: Perspectives on Gynecologic Care in African Immigrant and Refugee Women in Boston, Massachusetts.

African-born immigrant women, and particularly refugees and asylum seekers, are at risk for reproductive health disparities but inadequately use relevant gynecologic services. We sought to elucidate perspectives on gynecologic care in a population of Congolese and Somali immigrants. We conducted a secondary qualitative analysis of focus group data using a grounded theory approach and the Integrated Behavioral Model as our theoretical framework. Thirty one women participated in six focus groups. Participant beliefs included the states of pregnancy and/or pain as triggers for care, preferences included having female providers and those with familiarity with female genital cutting. Barriers included stigma, lack of partner support, and lack of resources to access care. Experiential attitudes, normative beliefs, and environmental constraints significantly mediated care preferences for/barriers to gynecologic health service utilization in this population. Centering of patient perspectives to adapt delivery of gynecologic care to immigrants and refugees may improve utilization and reduce disparities.

Bromocriptine inhibits pro-opiomelanocortin mRNA and ACTH precursor secretion in small cell lung cancer cell lines.

We have previously reported that a human small cell lung cancer (SCLC) cell line (COR L103) that expresses the proopiomelanocortin (POMC) gene and secretes ACTH precursor peptides is relatively resistant to glucocorticoid regulation. Using this model, we have now examined alternative regulatory mechanisms of the POMC gene and found that both the mRNA and ACTH precursor peptides were stimulated four- and two-fold, respectively, after 48 h incubation with db-cAMP. Next, we examined the dopamine agonist, bromocriptine, which acts predominantly through D2 receptors linked to adenyl cyclase to cause a reduction in intracellular cAMP. Bromocriptine suppressed cAMP levels and inhibited precursor peptide secretion within 24 h in a dose-dependent manner (0.15-15 microM). At the highest dose, peptide secretion was inhibited from 95 to 53 pmol/mg protein, and POMC mRNA was reduced by 50%, while beta-actin mRNA remained unchanged. This effect could not be mimicked by incubation of cells with the alpha-adrenergic antagonist, phenoxybenzamine, suggesting that the alpha-adrenergic effects of bromocriptine were not responsible for this observation. These cells also secrete estradiol, but the secretory rate was unaffected by bromocriptine, suggesting, with the beta-actin data, that the POMC inhibition was not a cytotoxic effect. No recovery in precursor peptide secretion was seen in a 48-h period after the removal of bromocriptine. However, when the postchallenge incubation was extended to 8 d, there was a recovery in secretory potential between day 3 and day 8 and normal growth kinetics in the 4 d after removal of the drug. In contrast to these findings, the mouse corticotroph cell line, AtT20, showed no response to bromocriptine, in keeping with reports that this agonist has no effect on anterior lobe corticotrophs. We conclude that bromocriptine effectively inhibits POMC expression in SCLC cells, and that this phenomenon might be of useful clinical application.

The early response gene NGFI-C encodes a zinc finger transcriptional activator and is a member of the GCGGGGGCG (GSG) element-binding protein family.

We have cloned NGFI-C, a nerve growth factor-induced early-response gene which encodes a Cys2/His2 zinc finger protein. RNA blot analysis demonstrates that NGFI-C mRNA is induced within minutes of stimulation of PC12 cells by nerve growth factor and is similarly activated in brain after a Metrazol-induced seizure. The cDNA sequence predicts a protein that contains three zinc fingers which show striking homology to the DNA-binding regions of three previously reported zinc finger proteins, NGFI-A, Krox-20, and the Wilms' tumor gene product. NGFI-C binds to the previously described DNA-binding site of these three proteins, which is GCGGGGGCG. Cotransfection experiments revealed that NGFI-C strongly activates transcription from this site in mammalian cells. The isolation of another early-response gene that encodes a member of the G(C/G)G or GSG element-binding family should provide an opportunity to investigate the relative contributions of a family of transcription factors to the cell's response to changes in its environment.

Pharmacokinetics of oral zidovudine (azidothymidine) in patients with AIDS when administered with and without a high-fat meal.

Zidovudine is the only drug currently approved for the treatment of HIV infection. The present recommended doses found to be efficacious in patients with AIDS (200 mg every 4 h) achieve serum zidovudine concentrations greater than 0.267 micrograms/ml (1 mumol/l). Since patients often take zidovudine with food, we have investigated the effect of a liquid high-fat meal on the rate of absorption of zidovudine and on the peak serum concentration achieved. Eight patients received their usual dose of zidovudine (100 mg or 250 mg), with and without a liquid high-fat meal, on two separate study days, in a randomized crossover fashion. Blood and urine samples were collected over a 4-h period. In the absence of food, zidovudine is rapidly absorbed; the time to reach maximal serum concentration (Tmax) was 0.68 (+/- 0.25) h and the mean peak serum concentration (Cmax) achieved was 0.49 (+/- 0.3) micrograms/ml (dose normalized to 100 mg dose). In the presence of a high-fat meal, Tmax was significantly prolonged [1.95 (+/- 0.69) h; P less than 0.05] and the Cmax reduced [0.245 (+/- 0.12) micrograms/ml; P less than 0.05]. This demonstrates that to achieve maximal zidovudine serum concentrations, patients should take this medication on an empty stomach.

Development of adrenocorticotropin-(1-39) and precursor peptide secretory responses in the fetal sheep during the last third of gestation.

Although it is known that concentrations of immunoreactive ACTH increase during late gestation in fetal sheep plasma, the nature of the ACTH has not been well characterized. We used two-site immunoradiometric assays to separately measure high mol wt ACTH precursors (POMC and pro-ACTH) and ACTH-(1-39) in plasma of fetal sheep with chronic arterial and venous catheters. We compared the ratio of these peptides as a function of gestational age under basal conditions and in response to exogenous vasopressin and/or corticotropin-releasing hormone. Under basal conditions, the concentration of precursors was not changed throughout the last third of gestation; however, ACTH-(1-39) increased significantly approaching term. The molar ratio of precursors to ACTH-(1-39), therefore, decreased from 15.8 +/- 1.0 at 110 days to 7.9 +/- 0.6 at 140 days gestation. At all gestational ages, vasopressin and corticotropin-releasing hormone increased ACTH-(1-39) and precursors, albeit with different time courses. At 120 days gestation, arginine vasopressin plus CRH produced synergistic increases in ACTH-(1-39) and precursors, whereas the response was only additive at other ages. The present results indicate that the elevation in the resting plasma immunoreactive ACTH concentration that occurs near term is constituted by an increase in the concentration of ACTH-(1-39) relative to those of POMC and pro-ACTH, which may have further physiological significance. Also, CRH and AVP are potent stimulators of both ACTH-(1-39) and ACTH precursors.

Direct measurement of the precursors of adrenocorticotropin in human plasma by two-site immunoradiometric assay.

An immunoradiometric assay (IRMA) for the direct measurement of the precursors of ACTH in unextracted human plasma has been developed and evaluated clinically in normal subjects and patients with disorders of the hypothalamic-pituitary-adrenal axis. The IRMA is based on an iodinated monoclonal antibody to ACTH and a monoclonal antibody to gamma MSH coupled to Sephacryl S300. The assay detects only peptides containing both epitopes, i.e. POMC (31K) and pro-ACTH (22K). The reference standard was partially purified POMC from culture medium of human corticotroph adenoma cells. The detection limit (greater than +2.5SD of the 0 standard) was 2.0 pmol/L and the within-assay coefficient of variation was less than 10% between 29 and 2600 pmol/L. Plasma concentrations of ACTH precursor peptides in 11 normal subjects sampled at 0930 h ranged from 5-34 pmol/L. The concentrations in the patient groups studied were: 260-2300 pmol/L in 5 patients with the ectopic ACTH syndrome associated with small cell lung cancer, less than 2.0-104 pmol/L in 10 patients with pituitary-dependent Cushing's disease, 23 pmol/L in a patient with Nelson's syndrome, and 3.0-230 pmol/L in 5 patients with Addison's disease. We conclude that this IRMA offers a simple and reliable method for measuring ACTH precursors in unextracted plasma. The proportionately greater elevation of ACTH precursors compared to ACTH in patients with the ectopic ACTH syndrome associated with small cell lung cancer but not in pituitary-dependent Cushing's syndrome, suggests that this assay may be clinically useful.

Differential release of proopiomelanocortin-derived peptides from the human pituitary: evidence from a panel of two-site immunoradiometric assays.

In humans, proopiomelanocortin (POMC) and the peptides derived from it have been individually identified in plasma under differing conditions. However, direct quantitative comparison has proved difficult because of the limitations of RIAs. Using a panel of monoclonal antibodies recognizing different regions of POMC, we have developed specific two-site immunoradiometric assays (IRMAs) for the ACTH precursors (POMC and pro-ACTH), ACTH, beta-lipotropin (beta LPH), beta-endorphin (beta EP), and the N-terminal POMC fragment (N-POC). We have quantified these peptides directly in plasma from normal subjects under basal conditions and in response to different regulatory factors. Basal levels of ACTH precursors, 5-40 pmol/L, were greater than or equal to ACTH, less than 0.9-11.3 pmol/L; N-POC, 5.6-16.8 pmol/L; beta LPH, 2.5-6.7 pmol/L; and beta EP less than or equal to 1.7 pmol/L. ACTH, N-POC, beta LPH, and beta EP levels increased in parallel in response to metyrapone (n = 8) and decreased in response to dexamethasone (n = 8), whereas ACTH precursor concentrations did not respond. After human CRH administration, peripheral concentrations of ACTH, N-POC, and beta LPH showed similar increments (median increment, 163%, 145%, and 172%, respectively; n = 6). POMC peptide responses to human CRH were also assessed in inferior petrosal sinuses draining the pituitary in 20 patients with pituitary-dependent Cushing's disease. In these patients, the increment in ACTH after CRH exceeded that in ACTH precursors by 4-fold (median, 459% and 96%). An increase in the ratios of ACTH/N-POC and ACTH/beta LPH was also apparent after CRH stimulation. The increment in beta EP after CRH always exceeded the increments in POMC and beta LPH. In summary, these data suggest that significant concentrations of ACTH precursors are present in the circulation of normal subjects, that ACTH precursors are not regulated in the same way as the processed POMC peptides, and that ACTH and beta EP are preferentially released from the pituitary in response to CRH.

Elevated levels of adrenocorticotropin (ACTH) precursors in post-adrenalectomy Cushing's disease and their regulation by glucocorticoids.

The ACTH precursor pro-opiomelanocortin (POMC) undergoes specific endoproteolytic cleavages in the anterior pituitary gland to give ACTH-(1-39). ACTH precursors circulate in normal subjects and are high both in the ectopic ACTH syndrome and in patients with aggressive pituitary adenomas. This study examines plasma levels of ACTH precursors and ACTH in 24 patients with post-adrenalectomy Cushing's disease, in 10 of whom computed tomography showed evidence of tumor progression. ACTH precursors were higher in post-adrenalectomy Cushing's disease (median 97.5 pmol/L, range 26-647 pmol/L) than in untreated Cushing's disease (median 29 pmol/L, range 9-104 pmol/L) and normal controls (5-40 pmol/L) (P < 0.001) and were significantly higher in patients with larger tumors (median 175 pmol/L, range 52-647 pmol/L) than in the remainder (median 41 pmol/L, range 26-510 pmol/L) (P = 0.02). Surprisingly, pro-opiomelanocortin (POMC) processing to ACTH was enhanced in post-adrenalectomy patients (ratio 1 +/- 0.5) compared with Cushing's disease (5.6 +/- 0.8) and normal subjects (5.3 +/- 0.9). After hydrocortisone ACTH precursors rose in 36% of post-adrenalectomy patients (increase 15-78%) and remained within 10% of basal levels in 46%. Six patients had a rise in precursors and a fall in ACTH suggesting differential regulation of these peptides. In conclusion, whereas ACTH precursors are high in post-adrenalectomy Cushing's disease and higher levels correlate with tumor progression, processing of precursors to ACTH is enhanced.

Proopiomelanocortin is the predominant adrenocorticotropin-related peptide in human cerebrospinal fluid.

High mol wt forms of immunoreactive ACTH and beta-endorphin (beta EP) are present in cerebrospinal fluid (CSF). We have quantified these peptides directly in the CSF of 26 patients undergoing routine myelography, using a panel of monoclonal antibody-based two-site immunoradiometric assays, specific for ACTH precursors (both POMC and pro-ACTH cross-react 100%), POMC, ACTH, and beta EP. The mean +/- SD levels of POMC in CSF were 530 +/- 150 pmol/L similar to total ACTH precursor immunoreactivity (414 +/- 83 pmol/L). By comparison, the CSF levels of ACTH (3.2 +/- 0.6 pmol/L) and beta EP (6.7 +/- 2.9 pmol/L) were 100-fold lower. POMC, by virtue of its 1% cross-reactivity in the ACTH immunoradiometric assay, could have also accounted for the ACTH immunoreactivity in CSF. Sephadex G-75 chromatography of CSF confirmed the presence of a single major peak of ACTH precursors eluting at the position of POMC (31K), while ACTH immunoreactivity was not detected at the position of ACTH-(1-39) (4.5K). We also studied the effect of exogenous glucocorticoids on CSF POMC peptides by giving 2.5 mg dexamethasone (0.5 mg, orally, every 6 h for 24 h) to a similar group of age-matched patients before lumbar puncture. No significant differences in CSF peptide content were observed between the two groups. These data suggest that the unprocessed precursor molecule POMC is the predominant peptide of the POMC family in human CSF and should always be considered when interpreting data involving ACTH or other component peptide immunoreactivity in this biological fluid.

Defective glucocorticoid regulation of proopiomelanocortin gene expression and peptide secretion in a small cell lung cancer cell line.

A human small cell lung cancer cell line (COR L103) that actively expresses the proopiomelanocortin (POMC) gene has been used as a model of extrapituitary ACTH-secreting tumors to investigate the phenomenon of resistence of ACTH production to glucocorticoids. After both short term (24 h) and long term (10 days) exposure to hydrocortisone at concentrations of 500 and 1000 nM, the accumulation of intracellular POMC mRNA, ACTH, and ACTH precursor peptides in the culture medium was not suppressed. These finding contrast with those in the pituitary corticotroph cell line AtT20, in which POMC mRNA, ACTH, and ACTH precursors were suppressed under the same conditions. Two other genes that are regulated by glucocorticoids in other cell types, the tyrosine amino transferase gene and the glucocorticoid receptor gene, were expressed in COR L103 cells. However, neither gene appeared to be regulated by hydrocortisone in this small cell lung cancer cell line. Further studies demonstrated that glucocorticoid receptor binding could be detected in the nucleus and cytoplasm, with a Kd of 5 X 10(-9) M. It is concluded that nonsuppression of POMC by glucocorticoids is probably part of a more global defect of glucocorticoid signaling in these cells, but that this defect lies distal to steroid binding in the nucleus.

Choice of treatment affects plasma levels of insulin-like growth factor-binding protein-1 in noninsulin-dependent diabetes mellitus.

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) modulates the metabolic and mitogenic effects of IGFs. Although IGFBP-1 levels are abnormally high in insulin-dependent diabetes (IDDM), relatively little is known in NIDDM; conflicting data have suggested both high and low levels. We investigated whether treatment modifies IGFBP-1 levels in two groups of NIDDM patients. Study 1 examined fasting concentrations in groups of patients with NIDDM, comparable except for treatment type (sulfonylurea, n = 23; once daily insulin, n = 15; sulfonylurea plus once daily insulin, n = 14; multiple insulin injections, n = 9) and 25 nondiabetic subjects. In sulfonylurea-treated patients there were markedly reduced plasma IGFBP-1 concentrations (median, interquartile range in parentheses): control, 61.0 (36-96) micrograms/L; sulfonylureas alone, 31.5 (21-61) micrograms/L (P < 0.01); and sulfonylureas plus insulin, 31.5 (9-53) micrograms/L (P < 0.01). Once daily insulin was associated with values similar to those in the control group [62.0 (27-103) micrograms/L; P = NS], whereas IGFBP-1 levels were higher with multiple insulin injection therapy [156.0 (71-184) micrograms/L; P < 0.05]. Proinsulin levels were higher in sulfonylurea-treated patients, but there was no significant correlation between IGFBP-1 and proinsulin within any individual group. Study 2 examined the effects of treatment on the dynamics of IGFBP-1 levels between 0800-1900 h. In control subjects (n = 8), levels fell from 0800 h (mean +/- SEM, 22.4 +/- 5.2 micrograms/L) to 1000 h (14 +/- 5.2 micrograms/L), followed by a rise, more rapid after food, to a peak at 1240 h (20.6 +/- 3.7 micrograms/L). Levels then declined until 1500 h (10.7 +/- 2.9 micrograms/L), with a further postprandial peak at 1840 h (23.1 +/- 3.2 micrograms/L). Sulfonylurea therapy (n = 6) resulted in a complete loss of this pattern, with a marked fall in IGFBP-1 from 0800 h (22 +/- 2.7 micrograms/L) to less than 7 micrograms/L for the remainder of the study (area under the curve, 1150-1400 h, P < 0.001 vs. control). By contrast, in metformin-treated patients (n = 7), neither IGFBP-1 levels nor postprandial peaks were significantly different from those in the control group. Our findings suggest that in patients with NIDDM, the regulation of IGFBP-1 is markedly influenced by the choice of treatment.

Impaired processing of proopiomelanocortin in corticotroph macroadenomas.

The regulation and secretion of the ACTH precursors POMC and pro-ACTH were assessed directly using a 2-site immunoradiometric assay in six patients with pituitary macroadenomas (> or = 1.2 cm in diameter) and 27 patients with Cushing's disease due to a microadenoma. ACTH precursor levels were elevated in patients with macroadenomas (150-3690 pmol/L; normal range, < 5-40 pmol/L) and significantly higher than those in microadenoma patients (median, 29 pmol/L; range, 9-104 pmol/L; P < 0.001). Patients with macroadenomas also had increased ACTH precursor/ACTH ratios (15-181:1) compared with microadenoma patients (median, 5:1, range, 0.7-18.5:1; P < 0.001). ACTH precursors were unresponsive to high dose dexamethasone in patients with macroadenomas, whereas ACTH and cortisol responses varied. After CRH administration, ACTH precursors were unchanged, whereas cortisol increased significantly, suggesting the release of biologically active ACTH. This study clearly demonstrates reduced processing of POMC to ACTH in large pituitary tumors, a characteristic usually associated with tumors causing the ectopic ACTH syndrome, and provides evidence for differential regulation of ACTH precursors and ACTH by glucocorticoid and CRH. Variation in the clinical symptoms of patients with corticotroph macroadenomas may be attributable to differences in biological potency between the ACTH precursors and ACTH.

Pro-opiomelanocortin gene expression and peptide secretion in human small-cell lung cancer cell lines.

Expression of the RNA coding for the ACTH-beta-lipotrophin precursor, pro-opiomelanocortin (POMC), has been demonstrated in five human small-cell lung cancer (SCLC) cell lines. Using Northern and slot-blot hybridization analysis of RNA and a bovine POMC cDNA as probe, the processed POMC RNA from SCLC cells was found to be approximately 1350 nucleotides in length, which is larger than that found in the normal human pituitary. Expression of the POMC gene was confirmed by measurement of ACTH precursors secreted by the cells, using a novel two-site immunoradiometric assay based on monoclonal antibodies, which directly quantifies both POMC and pro-ACTH but does not recognize ACTH. Levels of POMC in medium accumulated throughout the growth of the cells, in contrast to POMC RNA which showed a relatively constant level of expression. We conclude that human SCLC cell lines are valuable models for studying the aberrant expression and regulation of the human POMC gene.

Glucocorticoid inhibition of ACTH peptides: small cell lung cancer cell lines are more resistant than pituitary corticotroph adenoma cells.

In the normal pituitary, glucocorticoids are the principal negative regulatory of the pro-opiomelanocortin (POMC) gene which gives rise to the biologically active peptides ACTH and beta-endorphin. In Cushing's syndrome, ACTH-secreting pituitary tumours show a degree of glucocorticoid resistance, whilst ACTH-secreting extra-pituitary tumours have an even greater resistance to glucocorticoid excess. In an attempt to understand the mechanism of this phenomenon, we have compared the effects of glucocorticoids on POMC mRNA and peptide secretion in human and mouse corticotroph adenoma cells and in small cell lung carcinoma (SCLC) cells. ACTH precursor peptides were inhibited within 24 h by 25-50 nM hydrocortisone in primary cultures from a human corticotroph adenoma. In the mouse corticotroph adenoma cell line (AtT20), inhibition of both ACTH precursors and ACTH was not observed after 24 h but, by 10 days, glucocorticoids suppressed peptide levels with a concentration causing 50% inhibition of 50 nM hydrocortisone and maximal inhibition at 500 nM hydrocortisone. In marked contrast, there was no response to 500 nM hydrocortisone in the five SCLC cell lines (COR L103, COR L42, COR L24, COR L31, DMS 79) all of which secrete ACTH precursors. However, two of the five SCLC cell lines (COR L31 and DMS 79) were responsive to 1000 nM hydrocortisone. POMC mRNA, quantitated by slot-blot analysis, gave similar results for the five SCLC cell lines, implying that the abnormality may occur at the level of gene expression. When one of the three resistant cell lines (COR L103) was incubated with 2000 nM hydrocortisone or 2000 nM dexamethasone a clear suppression of precursor peptides and POMC mRNA was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of ACTH and ACTH precursor peptides secreted by human pituitary and lung tumour cells in vitro.

The molecular forms of ACTH secreted by established human small cell lung cancer (SCLC) cells and primary cultures derived from a bronchial carcinoid tumour, a pituitary adenoma and hyperplastic pituitary tissue have been characterized by Sephadex G-75 chromatography and quantified with two novel immunoradiometric assays for ACTH and ACTH precursor peptides. Pro-opiomelanocortin (POMC; Mr 31,000) and pro-ACTH (Mr 22,000) were secreted by all cell types. No smaller peptides were identified in the culture media from SCLC and bronchial carcinoid cells, implying a deficiency in the enzymes and/or intracellular organelles required for extensive POMC processing. A more heterogeneous profile of ACTH-containing peptides was produced by cells of pituitary origin, indicating more extensive proteolytic processing of POMC. However, the major peptide secreted by cells from a large aggressive pituitary adenoma was unprocessed POMC (Mr 31,000). These results suggest that both lung and pituitary cells in vitro retain their in-vivo pattern of POMC processing and provide valuable models in which to study the regulation of ACTH synthesis and secretion.

Increasing gestational age and cortisol alter the ratio of ACTH precursors:ACTH secreted from the anterior pituitary of the fetal sheep.

We have used a perifusion system and slices of the anterior pituitary of the fetal sheep combined with specific immunoradiometric assays to investigate the effect of increasing gestational age and cortisol infusion on the output of ACTH(1-39) and the ACTH precursors, pro-ACTH and pro-opiomelanocortin, from the fetal sheep pituitary. Two slices from each fetal anterior pituitary at 106-113 days (n = 3), 120-136 days (n = 5) and 140-143 days (n = 5) of gestation were used. Slices from each anterior pituitary were perifused with the perifusion buffer for at least 120 min prior to the infusion of cortisol (100 nM) for 30 min or buffer alone (control). The anterior pituitary output (fmol/5 min per mg pituitary) of ACTH(1-39) and the ACTH precursors were measured using specific immunoradiometric assays. There was a significant increase in the anterior pituitary secretion rate of ACTH(1-39) between 120 and 136 days (1.04 +/- 0.23 fmol/5 min per mg) and between 140 and 143 days of gestation (3.08 +/- 0.33 fmol/5 min per mg). In contrast, there was no change in the secretory rate of the ACTH precursors between 105 and 143 days of gestation. The ratio of the anterior pituitary output of the ACTH precursors:ACTH(1-39) therefore decreased between 120 and 143 of days gestation from 19.10 +/- 2.05 to 6.36 +/- 0.58. There was no effect of cortisol infusion on the anterior pituitary secretion of either ACTH(1-39) or the ACTH precursors before 116 days of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)