Changes in the phenotype of human small cell lung cancer cell lines after transfection and expression of the c-myc proto-oncogene.

Small cell lung cancer growing in cell culture possesses biologic properties that allow classification into two categories: classic and variant. Compared with classic small cell lung cancer cell lines, variant lines have altered large cell morphology, shorter doubling times, higher cloning efficiencies in soft agarose, and very low levels of L dopa decarboxylase production and bombesin-like immunoreactivity. C-myc is amplified and expressed in some small cell lung cancer cell lines and all c-myc amplified lines studied to date display the variant phenotype. To investigate if c-myc amplification and expression is responsible for the variant phenotype, a normal human c-myc gene was transfected into a cloned classic small cell lung cancer cell line not amplified for or expressing detectable c-myc messenger RNA (mRNA). Clones were isolated with one to six copies of c-myc stably integrated into DNA that expressed c-myc mRNA. In addition, one clone with an integrated neo gene but a deleted c-myc gene was isolated and in this case c-myc was not expressed. C-myc expression in transfected clones was associated with altered large cell morphology, a shorter doubling time, and increased cloning efficiency, but no difference in L dopa decarboxylase levels and bombesin-like immunoreactivity. We conclude increased c-myc expression observed here in transfected clones correlates with some of the phenotypic properties distinguishing c-myc amplified variants from unamplified classic small cell lung cancer lines.

Cerebrospinal fluid bombesin and calcitonin in patients with central nervous system metastases from small-cell lung cancer.

To determine whether levels of mammalian bombesin (MB) or calcitonin would be useful in detecting CNS metastases in patients with small-cell lung cancer (SCLC), we measured their concentrations in the CSF of 94 patients who underwent lumbar puncture for suspected CNS involvement. The MB concentration was significantly elevated in the 51 patients with definite CNS metastases as compared with the 30 patients without apparent CNS involvement (P less than .01). This significance was due to increased levels of MB in 18 patients with meningeal carcinomatosis. Whereas CSF MB was detectable (greater than 10 fmol/mL) in only 7% of patients without apparent CNS involvement, CSF MB was detectable in 21% with parenchymal CNS metastases and in 78% of those with meningeal carcinomatosis. Interestingly, 93% of patients having MB concentrations above 20 fmol/mL had meningeal metastases. The calcitonin concentration was significantly elevated in 42 patients with CNS metastases as compared with 27 patients without CNS involvement (P less than .01). Both the 15 patients with meningeal carcinomatosis and the 27 patients with only parenchymal metastases had significantly elevated levels of CSF calcitonin as compared with those without CNS metastases. Fifty-three percent of patients with meningeal carcinomatosis and 48% with parenchymal metastases had a CSF calcitonin level above 18 fmol/mL, whereas only 7% without apparent CNS metastases exceeded this level. Sixty-seven percent of all patients with CNS metastases had increased CSF levels of one of the two hormonal markers. Thus, in SCLC patients, an elevated CSF calcitonin strongly suggested CNS metastases and an elevated MB was very suggestive of the presence of meningeal carcinomatosis. However, only the latter observation seems of clinical importance due to the difficulties in establishing this diagnosis with current diagnostic measures.

Ubiquitous expression of the calcitonin-i gene in multiple tissues in response to sepsis.

Calcitonin precursors (CTpr), including procalcitonin, are important markers and also potentially harmful mediators in response to microbial infections. The source and function of CTpr production in sepsis, however, remains an enigma. In the classical view, the transcription of the CT-I gene is restricted to neuroendocrine cells, in particular the C cells of the thyroid. To better understand the pathophysiology of CTpr induction in sepsis, we used an animal model analog to human sepsis, in which bacterial infection is induced in hamsters by implanting Escherichia coli pellets ip. Compared with control hamsters, levels of CTpr were elevated several fold in septic plasma and in nearly all septic hamster tissues analyzed. Unexpectedly, CT-messenger RNA was ubiquitously and uniformly expressed in multiple tissues throughout the body in response to sepsis. Notably, the transcriptional expression of CT-messenger RNA seemed more widely up-regulated in sepsis than were classical cytokines (e.g. tumor necrosis factor-alpha and interleukin-6). Our findings, which describe a potentially new mechanism of host response to a microbial infection mediated by CTpr, introduce a new pathophysiological role for the CT-I gene.

Serum calcitonin precursors in sepsis and systemic inflammation.

High serum levels of the calcitonin (CT) prohormone, procalcitonin (pro-CT), and its component peptides occur in systemic inflammation and sepsis. Using two different assays, we undertook a prospective study to determine the utility of serum precalcitonin peptides (pre-CT) as markers in this condition. Twenty-nine patients meeting criteria for the systemic inflammatory response syndrome were studied daily in two intensive care units. Sera were collected, and APACHE II scores were determined until recovery or death. All patients had markedly elevated serum pre-CT. Prognostically, peak values were the most important. The highest values portended mortality, and a lower level could be ascertained below which all patients survived. Peak pre-CT levels were significantly higher in patients with infection documented by blood cultures than in those patients with no documented infection from any source (P < 0.05). Mature CT remained normal or only moderately elevated. Compared with the serum pre-CT levels, receiver operating characteristic curve analysis revealed that the APACHE II scores, although more cumbersome, were better overall predictors of mortality. Thus, pre-CT is an important serum marker for systemic inflammatory response syndrome and is predictive of outcome. It also provides data concerning the presence of severe infection and may prove to be clinically useful for proactive patient care.

Immunoneutralization of procalcitonin as therapy of sepsis.

Prior studies have demonstrated that the prohormone, procalcitonin (ProCT), and its component calcitonin precursors (CTpr) are increased in the serum of septic patients, correlate with the severity of the illness, and persist for relatively long periods of time. Animal studies in septic hamsters have revealed that the administration of ProCT is toxic and that immunoneutralization with IgG that is reactive to this molecule significantly improves survival. A large animal model of a very rapidly lethal polymicrobial sepsis has been developed in the pig in order to measure continuous physiological and metabolic parameters and also to compare the effects in this animal of an immunoneutralization, which is performed late in the course of the disease, to an identical, but early, therapy. Based upon the physiological and metabolic parameters, the late therapy, which was initiated during the fourth hour at a time when pigs were nearly moribund, was found to be as beneficial as early therapy. In both late and early therapy, the only animals to survive at the predetermined time of euthanasia were those which had received immunoneutralization therapy.

Small cell lung carcinoma cell lines express mRNA for calcitonin and alpha- and beta-calcitonin gene related peptides.

Calcitonin (CT) and calcitonin gene related peptide (CGRP) are derived from preprohormones encoded by three mRNAs (CT, alpha-CGRP and beta-CGRP) from two genes (CALC1 and CALC2) on chromosome 11. Among 16 small cell lung cancer cell lines examined by RNase protection assay, 9 (56%) had detectable CT mRNA, 8 (50%) had alpha-CGRP mRNA, and 13 (81%) had beta-CGRP mRNA. At least one CALC1 transcript (CT or alpha-CGRP) was found in 11 (69%) cell lines with three having only CT mRNA, two having only alpha-CGRP mRNA, and six having both. beta-CGRP mRNA was detected in all of these 11 cell lines expressing a CALC1 transcript. Immunoreactive CT was detected by radioimmunoassay in eight of nine SCLC cell lines expressing CT mRNA, and immunoreactive CGRP was detected in 12 of 13 cell lines expressing a CGRP mRNA. The variety of expression of these three peptides in different cell lines of the same cell type should provide a useful system for further study of the control of expression of these peptides.

Calcitonin secretion by continuous cultures of small cell carcinoma of the lung: incidence and immunoheterogeneity studies.

A study was made of immunoreactive calcitonin (iCT) secretion by continuous cultures of small cell carcinoma of the lung (SCCL). Using an antiserum region specific for the midportion of the molecule, 9/12 cultures were found to secrete iCT. Gel filtration studies were performed on both supernatant fluid (SF) and cell pellet (CP) extract from a culture secreting high levels of iCT. Multiple iCT fractions were found in the SF with the major fraction being of high molecular weight (MW). In contrast, the CP had apparently monomeric CT as its principal iCT fraction. These studies demonstrate frequent iCT secretion by SCCL cultures and significant disparities between the iCT moieties found extra- and intracellularly.

Procalcitonin and proinflammatory cytokine interactions in sepsis.

Immunoneutralization of procalcitonin (ProCT), a putative mediator of sepsis, has been shown to increase survival in an animal model of sepsis. To better understand the role that ProCT plays in the sepsis cascade, we studied the relationship of this hormone to the proximal proinflammatory mediators, IL-1beta and TNFalpha. Hamsters were made septic by i.p. implantation of Escherichia coli-impregnated agar pellets. A time line study of serum IL-beta, TNFalpha, and ProCT levels showed that the increase in the cytokines was transient and less than 2-fold over baseline, whereas ProCT increased >100-fold by 12 h and remains elevated through 24 h. TNFalpha (400 microg/kg) was injected into healthy animals, inducing an elevation in ProCT that was 25-fold greater than controls. ProCT (30 microg/kg) was given to healthy and septic animals. In healthy animals, there was no significant elevation in serum IL-1beta or TNFalpha levels. In septic animals, IL-1beta was modestly blunted at 3 h but not at 12 h, and there was no change in TNFalpha levels. ProCT did not initiate or enhance IL-1beta or TNFalpha expression; however, the massive and sustained elevation of this hormone seen in sepsis can be induced by the proximal cytokine, TNFalpha. This study suggests that ProCT is a secondary mediator that might augment and amplify but does not initiate the septic response. Immunoneutralization of ProCT may prove to be an important clinical strategy, in view of its sustained elevation and the difficulty in initiating therapy for sepsis during the early phases of illness.

Procalcitonin and proinflammatory cytokine in interactions in sepsis.

Immunoneutralization of procalcitonin (ProCT), a putative mediator of sepsis, has been shown to increase survival in an animal model of sepsis. To better understand the role that ProCT plays in the sepsis cascade, we studied the relationship of this hormone to the proximal proinflammatory mediators, IL-1beta and TNFalpha. Hamsters were made septic by i.p. implantation of Escherichia coli-impregnated agar pellets. A time line study of serum IL-beta, TNFalpha, and ProCT levels showed that the increase in the cytokines was transient and less than 2-fold over baseline, whereas ProCT increased >100-fold by 12 h and remains elevated through 24 h. TNFalpha (400 microg/kg) was injected into healthy animals, inducing an elevation in ProCT that was 25-fold greater than controls. ProCT (30 microg/kg) was given to healthy and septic animals. In healthy animals, there was no significant elevation in serum IL-1beta or TNFalpha levels. In septic animals, IL-1beta was modestly blunted at 3 h but not at 12 h, and there was no change in TNFalpha levels. ProCT did not initiate or enhance IL-1beta or TNFalpha expression; however, the massive and sustained elevation of this hormone seen in sepsis can be induced by the proximal cytokine, TNFalpha. This study suggests that ProCT is a secondary mediator that might augment and amplify but does not initiate the septic response. Immunoneutralization of ProCT may prove to be an important clinical strategy, in view of its sustained elevation and the difficulty in initiating therapy for sepsis during the early phases of illness.

Increased serum calcitonin levels in bronchogenic cancer.

A study of 26 men with bronchogenic cancer demonstrated high serum calcitonin levels in 62 percent (16). Levels were particularly high in patients with small-cell cancer and adenocarcinoma. Two varieties of hypercalcitonemia have been encountered: (1) ectopic hypercalcitonemia, in which the hormone is secreted by the tumor, and (2) thyroidal hypercalcitonemia, in which the high values emanate from the thyroid gland. In several patients, serum calcitonin levels decreased following therapy for the cancer. Further studies are needed to evaluate the diagnostic value and clnical utility of serum calcitonin levels as a marker substance in bronchogenic cancer.

Increased serum and urinary calcitonin levels in patients with pulmonary disease.

Serum and urinary calcitonin levels were measured in patients with acute and chronic inflammatory diseases of the lung. Using both carboxyl terminal and midportion antisera, the incidence of increased immunoreactive values of this hormone was 68 percent for patients with emphysema, 59 percent for tuberculosis, and 89 percent for acute bacterial pneumonitis. In order to determine the source of the high levels of calcitonin, immunoperoxidase stains were made of sections of human lung; the hormone was found within the bronchial Kultschitzky cell (K cell). This suggests a specific endocrine role for the K cell, and may explain not only the high calcitonin levels in patients with inflammatory lung disease, but also the high levels associated with both carcinoid tumor and small cell carcinoma, which may originate from K cells. It is apparent that moderately high levels of calcitonin in a patient with pulmonary disease cannot always be associated with tumor.

Comparative studies of hamster calcitonin from pulmonary endocrine cells in vitro.

The lung-associated peptide calcitonin (CT) has been localized by immunocytochemical means to discrete pulmonary endocrine (PE) cells. A long-term cell culture of CT-staining PE cells has been established. The molecular configuration of immunoreactive (iCT) from PE cell extracts was determined by gel chromatography, revealing predominantly large molecular weight forms of iCT. The size distribution characteristics of PE Cell iCT were similar to those of intact hamster lung. In contrast, hamster thyroid extracts contain predominantly 4000 dalton iCT (presumed monomer) and apparent iCT fragments. The culture media of the PE cells were found to contain mainly 4000 dalton iCT. We conclude that although the predominant forms of iCT found within cultured PE cells are distinct from those found within thyroidal C-cells, both iCT producing cells release mainly the monomer into the media. Malignant human bronchial carcinoid cells store predominantly monomeric iCT while secreting large molecular weight forms of iCT. Since the PE cell is the putative precursor cell to neuroendocrine malignancies, the disparity noted in the processing of CT may have significant pathobiological implications.

Immunoreactive calcitonin in cerebrospinal fluid of man.

We have detected immunoreactive calcitonin (iCT) in the cerebrospinal fluid (CSF) of normal individuals. Using an antibody with midportion recognition, the mean +/- S.D. of the cerebrospinal iCT in 27 normal subjects was 28 +/- 14 pg/ml. The mean serum iCT was 89 +/- 68 pg/ml, the CSF/serum distribution ratio being 0.31. There were no significant correlations between CSF iCT or serum iCT and the calcium, magnesium, phosphate, sodium, potassium or chloride in the CSF or serum. Although there was a trend for serum iCT values to be related to CSF iCT values, it did not attain statistical significance. The demonstration that the CSF contains iCT may have important physiologic implications, and its measurement offers a useful parameter to study its effects on calcium metabolism and/or other aspects of brain function.

Immunochemical heterogeneity of calcitonin in man: effect on radioimmunoassay.

Determinations of blood levels of human calcitonin by radioimmunoassay have varied considerably in different laboratories. Much of the controversy over calcitonin levels can be attributed to the multiplicity of immunoreactive forms of the hormone (iCT), the differing region specificities of the antisera utilized for measurement by radioimmunoassay, protein effects, different rates of degradation of the various iCT fractions and the specific methodology of the radioimmunoassay.

Calcitonin precursor levels in human medullary thyroid carcinoma.

DESIGN: The hormonal serum marker for the presence and course of patients with medullary thyroid cancer (MTC) is the mature calcitonin (CT) peptide. Other CALC-1 gene products such as the 116-amino acid polypeptide prohormone, procalcitonin, as well as its component calcitonin precursors (CTpr) may also be increased in their sera. We performed a study to evaluate the clinical utility of serum levels CTpr in these patients. METHODS: Twenty-one patients with MTC (9 males, 12 females; 23-76 years of age) were evaluated. The diagnosis was confirmed by histologic examination, except for 2 (a proven RET mutation plus an abnormal pentagastrin-stimulated CT level). Nine patients had postoperative hypercalcitoninemia and 3 of these died. The specific assay for mature CT was a commercial immunoradiometric assay (hCT-IRMA); the immunoluminometric assay for CTpr (B.R.A.H.M.S Diagnostica, Berlin, Germany) detects intact procalcitonin and the free CT:CT carboxypeptide-1. RESULTS: All patients had detectable serum CTpr. These levels considerably exceeded those of mature CT, averaging 7.6-fold greater. CTpr levels correlated positively with mature CT (r = 0.61; p < 0.001). After pentagastrin administration, there was a parallelism of response between the two assays. Whenever there were known metastases, CTpr increased markedly. CONCLUSION: This study demonstrates the universal presence of CTpr in the blood of patients with MTC. The measurement of these peptides may offer a new dimension to the clinical evaluation of this malignancy.

Modulation of pulmonary bombesin by nicotine and vagotomy.

In pregnant hamsters, three transplacental injections of the ganglionic agonist nicotine resulted in a dose-dependent decrease in the concentration of mammalian bombesin (MB) in the lungs of neonatal (1 day old) animals. This decrease in neonatal MB did not occur if nicotine was given only once during gestation, or when it was given three times in conjunction with the ganglionic antagonist mecamylamine. In one week old animals born of mothers who had been exposed to three doses of nicotine during gestation, lung MB had returned to control levels. When nicotine was injected into neonatal animals, lung MB acutely increased. Right sided vagotomy to young hamsters resulted in an increase in the ratio of lung MB (right vs. left lobe) 1 week after surgery. Administration of nicotine to vagotomized animals resulted in decreased total lung MB and normalization of the MB ratio. Thus, nicotine has a potent modulatory influence on lung MB during fetal and neonatal development and maturation. This influence is also present in young animals that are subjected to partial denervation. Our hypothesis is that the innervation of pulmonary neuroendocrine (PNE) cells influences both PNE cell growth and its synthetic function. PNE MB, which is an epithelial and neoplastic growth factor, may play a role in this response.

Evidence for cigarette smoke-induced calcitonin secretion from lungs of man and hamster.

In hamsters, acute cigarette smoke inhalation increased serum levels of the hormone calcitonin; and, in humans, smoking of two high-nicotine content cigarettes increased serum and urine levels of this hormone. The source of this immunoreactive calcitonin (iCT) does not appear to be the thyroid gland, since previously thyroidectomized patients demonstrated a similar response. In the hamster, the increased serum iCT levels were accompanied by a decreased lung tissue iCT content and hypocalcemia. It is suggested that the source of the cigarette smoke-induced hypercalcitonemia is the lung, possibly from the iCT-containing pulmonary neuroendocrine (PNE) cells. Moreover, this response appears to be dependent on the nicotine content of the cigarettes.

Late pulmonary sequela following burns: persistence of hyperprocalcitonemia using a 1-57 amino acid N-terminal flanking peptide assay.

Seven patients were evaluated at a mean duration of 8.4 yr after sustaining inhalational injury associated with burns. At the time of re-examination, the patients were asymptomatic and had normal chest X-rays, and arterial blood gases. Three of the seven patients had abnormally elevated serum calcitonin levels. The spirometry (FEV1) measurements showed an inverse trend to that of the serum calcitonin levels. The elevated calcitonin levels had an abnormal predominance of the procalcitonin component as assessed by several region specific antisera. The serum calcitonin also showed a significant correlation with the hormone level which had been obtained at the time of prior discharge from the hospital (r = 0.91). Although there appears to be no or minimal chronic pulmonary sequela to inhalational injury in burns by pulmonary testing, we speculate that the hyperprocalcitonemia in some of the patients may reflect a long-term hyperplastic response of the bronchio-epithelial pulmonary neuroendocrine cells. The potential significance of this and other lung-associated endocrine markers is discussed.

Procalcitonin and its component peptides in systemic inflammation: immunochemical characterization.

BACKGROUND: The systemic inflammatory response syndrome (SIRS) is a marked, generalized response to a variety of injuries, if infection is implicated, the term "sepsis" is used. Systemic inflammatory response syndrome/sepsis, which is initiated by proinflammatory cytokines, has been found to be associated with increased serum levels of the prohormone of calcitonin, procalcitonin (ProCT) and its aminoterminus peptide (nProCT). The serum levels of ProCT and nProCT are very useful markers for SIRS/sepsis, and may be used to follow the course, the response to therapy, and/or the prognosis. We studied the serum levels and distribution of ProCT and its component peptides in normal persons for comparison with similar immunochemical and separatory studies in patients with neuroendocrine cancer and with SIRS/sepsis of various etiologies. METHODS: We studied pooled and extracted serum of 13 normal subjects, and sera of patients with neuroendocrine cancer and SIRS/sepsis, using region-specific immunoassays, gel filtration, and high performance liquid chromatography. RESULTS: Normal sera contained small but measurable levels of the intact ProCT molecule, nProCT, a conjoined calcitonin-calcitonin carboxyterminal peptide (CT:CCP-I), CCP-I, free mature CT, and calcitonin gene-related peptide (CGRP). Sera from neuroendocrine cancer usually contained high levels of these peptides. In such cases, free mature CT was always increased, the mean ratio of the intact ProCT to free CT being 168 +/- 68. Gel filtration and HPLC studies of patients with SIRS/sepsis revealed markedly increased levels of ProCT, nProCT, and CT:CCP-I in varying proportions. Mature CT was normal to minimally elevated. The ratio of ProCT to free CT was 2,900 +/- 800. Although serum CGRP is commonly increased in neuroendocrine cancer, it was very low or undetectable in SIRS/sepsis. CONCLUSIONS: These studies indicate that ProCT and its component peptides circulate in normal persons. The serum of patients with SIRS/sepsis contains greatly increased levels of ProCT, nProCT and often, CT:CCP-I. However, in this condition, post-translational processing is incomplete, resulting in mature CT levels that are normal or minimally elevated. In contrast, patients with neuroendocrine cancer have considerably high mature CT levels. Interestingly, although serum CGRP levels often are high in neuroendocrine cancer, they are low in SIRS/sepsis. The marked hyperprocalcitonemia of SIRS/sepsis is probably a consequence of the pro-inflammatory cytokine cascade, and appears to be secreted in a constitutive fashion; the cell(s) of origin of this remarkable hypersecretion is unknown. There is a very marked positive correlation between serum levels of ProCT and nProCT, and the lower level of sensitivity for nProCT may make its measurement a more useful marker for early or mild SIRS/sepsis.

Calcitonin gene-related peptide expression in sepsis: postulation of microbial infection-specific response elements within the calcitonin I gene promoter.

BACKGROUND: Recently, we reported an unexpected ubiquitous expression of calcitonin (CT)-mRNA in a hamster peritonitis model of sepsis. Using this animal model,we undertook a study to further investigate the pattern of expression of the calcitonin I (CALC-I) gene and CT gene-related peptide (CGRP)-mRNA in sepsis. METHODS: Live Escherichia coli impregnated in agar pellets were implanted in the peritoneal cavities of hamsters. Twelve hours after sepsis induction, the septic and healthy control animals were sacrificed and tissues and peritoneal macrophages were collected. CGRP-mRNA content was evaluated by reverse transcription polymerase chain reaction (RT-PCR), quantitated by the Taq-Man technique, and compared with the mRNA expression of CT, tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6). The 5' untranslated regions of the mRNA and potential alternative splicing sites were identified by 5' rapid amplification of cDNA ends. RESULTS: We found a tissue-wide, ubiquitous and uniform expression of CGRP-mRNA in all septic tissues examined. CGRP-mRNA was detectable by RT-PCR in various extraneuronal and extrathyroidal septic tissues, but not in healthy control tissues. As found for CT-mRNA in our earlier studies, CGRP-mRNA seemed to be more specifically up-regulated as compared with other classical cytokines (ie, II-6 and TNF-alpha). Importantly, the 5' untranslated sequence in control and septic thyroid was similar to the sequence obtained from septic spleen. CONCLUSIONS: We postulate the presence of microbial infection-specific response elements in the CALC-I gene promotor, which, upon a specific stimulus, override the tissue-selective expression pattern. This new form of endocrine plasticity may be of importance in the response to systemic inflammation.