Increased Rho-kinase expression and activity and pulmonary endothelial dysfunction in smokers with normal lung function.

Endothelial dysfunction is one of the main consequences of the toxic effects of cigarette smoke on the vascular system. Increasing evidence suggests that the small G-protein RhoA and its downstream effectors, the Rho-kinases (ROCKs), are involved in systemic endothelial dysfunction induced by cigarette smoke. This study aimed to evaluate the role of the RhoA/ROCKs pathway in pulmonary artery endothelial function in current smokers with normal lung function. Lung tissues were obtained from nonsmokers and smokers who underwent lobectomy for lung carcinoma. Arterial relaxation in response to acetylcholine (ACh) was assessed in isolated pulmonary arterial rings. Protein expressions and activities of endothelial nitric oxide synthase (eNOS), ROCKs and the myosin phosphatase subunit 1 (MYPT-1) were sought. Relaxation in response to ACh was significantly lower in smokers as compared with nonsmokers (n = 8 in each group), consistent with reduced eNOS activity in the former compared with the latter. eNOS protein expression remained, however, the same in both groups. Expression of ROCKs, guanosine triphosphate-RhoA and phosphorylated MYPT-1 were significantly increased in smokers compared with controls. Pulmonary endothelial dysfunction is present in smokers whose lung function has not yet been impaired. Reduced activity of eNOS accounts at least in part for this endothelial dysfunction. Increased expression and activity of ROCKs accounts for another part through direct or indirect inhibition of the Rho-A/ROCKs pathway on nitric oxide synthesis and sustained pulmonary vasoconstriction through inhibition of myosin phosphatase.

Relationship of p21-activated kinase (PAK) and filopodia to persistence and oncogenic transformation.

Previously, we found that oncogenically transformed cells had fewer filopodia and more large, p21-activated kinase (PAK)-dependent features than normal cells. These large protrusions (LPs) were increased in cells expressing RhoA(N19) with Cdc42-associated kinase (ACK). Here, we determine how GTPase-mediated mechanisms of focal contact (FC) regulation affect these protrusions. Constructs encoding various proteins were introduced into cells which were then studied by microscopy and computerized image processing and analysis. Constructs that prevented PAK recruitment by PAK-interacting exchange factor (PIX) or restricted PAK residence time on FCs decreased both protrusions. Thus, filopodia were also PAK-dependent. A comparison of FC distribution in cells expressing PAK in the presence or absence of PAK kinase inhibitor domain (KID) suggested that PAK enlarged FCs without affecting the prevalence of either protrusion. KID or Nck expression increased LPs but not filopodia. Nck failed to synergize with KID or ACK and RhoA(N19) in enhancing LPs. Nck and KID synergistically enhanced filopodia, possibly because Nck recruited PAK to FCs while KID prevented their dissociation by PAK-mediated autophosphorylation. Coexpression of Nck, ACK, and RhoA(N19) abrogated filopodia and replicated the transformed phenotype. Since Nck recruitment of PAK is implicated in persistence of directional movement, we studied the PAK-Nck interface. Filopodia were eliminated by the Nck PAK-binding domain and LPs by the PAK Nck-binding domain. The results suggested that filopodia formation has more stringent requirements than LP formation, and Nck and PAK are used differently in the protrusions. Loss of filopodia in transformed cells may reflect defective regulation of GTPase mechanisms.

Immunolocalization of elastase in human emphysematous lungs.

The current working hypothesis concerning the pathogenesis of human pulmonary emphysema proposes that neutrophils migrate through the alveolar interstitium and degranulate, releasing proteolytic enzymes into the interstitium. These enzymes, in particular elastase, can bind to and degrade interstitial elastin. This report describes an immunohistochemical, ultrastructural technique that utilizes polyclonal antibodies to localize neutrophil elastase in human lungs. Using both the immunoperoxidase and the immunogold methods on thin, embedded sections of surgically resected human emphysematous lung tissue, elastase was localized in neutrophils in the lung interstitium and extracellularly in association with interstitial elastic fibers in human lungs that showed local emphysema of varying severity. Quantitative morphometric data were obtained from the lungs of eight patients undergoing lobectomy for removal of pulmonary carcinomas. Patients had preoperative forced expiratory volume (FEV1)% levels ranging from 55 to 77. There was a correlation between a quantitative measure of the local distribution of neutrophil elastase in contact with alveolar interstitial elastin and the local presence of emphysematous change as determined by mean linear intercept of the various histologic sections. These data support the validity of the "protease-protease inhibitor balance hypothesis" as an explanation of the pathogenesis of human pulmonary emphysema.

Interrelationships between the human alveolar macrophage and alpha-1-antitrypsin.

Alveolar macrophages lavaged from human lungs contain protease activity at an optimum pH of 3.0 and possibly a lesser peak of activity at pH 5.5. Protease activity measured at pH 4.1 is inhibited by purified alpha-1-antitrypsin. Fluorescent antibody studies of human alveolar macrophages showed that alpha-1-antitrypsin is present in normal alveolar macrophages. In addition, macrophages from a patient with a homozygous deficiency of alpha-1-antitrypsin exhibited less fluorescence when incubated in autologous serum than the same macrophages incubated in normal serum. Macrophages from normal subjects showed maximal fluorescence when removed from the lung and additional incubation with serum did not increase fluorescence. These results implicate the human alveolar macrophage as a possible source of an enzyme that may cause emphysema in patients deficient in alpha-1-antitrypsin. They also show that alpha-1-antitrypsin has access to the alveolus in normal subjects.

Invasive and metastatic potential of a v-Ha-ras-transformed human bronchial epithelial cell line.

The in vivo growth behavior and invasive potential of normal and "immortalized" human bronchial epithelial cells were studied by xenotransplantation procedures, an in vitro assay of invasiveness, and determinations of type IV collagenase activity and mRNA expression. BEAS-2B cells, immortalized after hybrid virus infection (adenovirus 12-simian virus 40), reconstituted a columnar epithelium when xenotransplanted into de-epithelialized rat tracheas transplanted sc into athymic BALB/c mice. A few adenomatous growths could be seen 16 weeks after transplantation. BZR cells, obtained by transfer of the v-Ha-ras oncogene into BEAS-2B cells, were tumorigenic in this xenotransplantation model. BZR-T33 cells, obtained from a tumor produced after injection of BZR cells, were also tumorigenic; however, they exhibited a shorter latent period. When these same cell lines were injected sc and iv into athymic BALB/c mice, BEAS-2B cells were not tumorigenic, and the BZR-T33 cells were more tumorigenic than the BZR cells. The incidence of spontaneous metastases after sc inoculation was zero for BEAS-2B cells, 33% for BZR cells, and 100% for BZR-T33 cells. Similar increasing values that correlated well with the data on in vivo growth were noted in the in vitro invasion assay, the collagenolytic ability, and the mRNA expression of type IV collagenase. Normal human bronchial epithelial cells showed the lowest values in all the assays. These progressive changes occurring in cells derived from the same parental line indicate that the presence of the v-Ha-ras oncogene in immortalized bronchial cells is associated with a full-fledged malignant phenotype, which is further enhanced by in vivo passaging.

Normal bronchial epithelial cell expression of glutathione transferase P1, glutathione transferase M3, and glutathione peroxidase is low in subjects with bronchogenic carcinoma.

Normal bronchial epithelial cells (NBECs) are at risk for damage from inhaled and endogenous oxidative species and from epoxide metabolites of inhaled polycyclic aromatic hydrocarbons. Epidemiological and in vitro data suggest that interindividual variation in this risk may result from variation in NBEC expression of enzymes that inactivate reactive species by conjugating them to glutathione. Quantitative competitive reverse transcription-PCR was used to measure mRNA levels of glutathione transferases (GSTs) and glutathione peroxidases (GSHPxs) in primary NBECs from subjects with or without bronchogenic carcinoma. Mean expression levels (mRNA/10(3) beta-actin mRNA) in NBECs from 23 subjects without bronchogenic carcinoma compared to those from 11 subjects with bronchogenic carcinoma respectively (in parentheses) were: mGST (26.0, 6.11), GSTM3 (0.29, 0.09), combined GSTM1,2,4,5 (0.98, 0.60), GSTT1 (0.84, 0.76), GSTP1 (287, 110), GSHPx (140, 62.1), and GSHPxA (0.43, 0.34). Levels of GSTP1, GSTM3, and GSHPx were significantly (P < 0.05) lower in NBECs from subjects with bronchogenic carcinoma. Further, the gene expression index formed by multiplying the values for mGST x GSTM3 x GSHPx x GSHPxA x GSTP1 had a sensitivity (90%) and specificity (76%) for detecting NBECs from bronchogenic carcinoma subjects that was better than any individual gene. In cultured NBECs derived from eight individuals without bronchogenic carcinoma and incubated under identical conditions such that environmental effects were minimized, the mean level of expression and degree of interindividual variation for each gene evaluated was less than that observed in primary NBECs. Data from these studies support the hypotheses that (a) interindividual variation in risk for bronchogenic carcinoma results in part from interindividual variation in NBEC expression of antioxidant genes; (b) gene expression indices will better identify individuals at risk for bronchogenic carcinoma than individual gene expression values; and (c) both hereditary and environmental exposures contribute to the level of and interindividual variation in gene expression observed in primary NBECs. Many epidemiological studies have been designed to evaluate risk associated with polymorphisms or gene expression levels of putative susceptibility genes based on measurements in surrogate tissues, such as peripheral blood lymphocytes. Based on data presented here, it will be important to include the assessment of NBECs in future studies. Measurement of antioxidant gene expression in NBECs may identify the 5-10% of individuals at risk for bronchogenic carcinoma. Bronchoscopic sampling of NBECs from smokers and ex-smokers then will allow susceptible individuals to be entered into surveillance and/or chemoprevention studies.

Lysyl oxidase expression in bronchogenic carcinoma.

BACKGROUND: Lysyl oxidase catalyzes a key step in the cross-linking of collagen and elastin in the extracellular matrix. Recent studies have documented differential lysyl oxidase expression in the stromal reaction to colon, breast, prostate, and lung cancer. The present study was undertaken to test the hypothesis that lysyl oxidase mRNA and protein expression decrease with advancing tumor stage in patients with bronchogenic carcinoma. METHODS: Tumor specimens were obtained from 17 patients undergoing resection for bronchogenic carcinoma. Real-time polymerase chain reaction was used to determine steady-state lysyl oxidase mRNA expression, and protein expression was qualitatively assessed by immunohistochemistry. RESULTS: Real-time polymerase chain reaction studies documented a 3.4-fold graded decrease in lysyl oxidase mRNA levels as tumors progressed from stage I to IV. Similar qualitative changes in lysyl oxidase protein expression were demonstrated by immunohistochemistry. CONCLUSIONS: These results support the hypothesis that variations in lysyl oxidase expression may correlate with the invasive and metastatic potential of bronchogenic carcinoma.

Debrisoquin oxidation genotype and susceptibility to lung cancer.

The association between the polymorphism of the cytochrome P450 debrisoquin hydroxylase (CYP2D6) and lung cancer is controversial. Previous reports suggested a link between CYP2D6 phenotype and lung cancer, with poor metabolizers having reduced susceptibility. Nevertheless, negative findings have also been published. By using allele-specific amplification, we have studied the frequency of four CYP2D6 (wild type and mutant) alleles in 89 patients with histologically proved bronchogenic carcinoma and in 98 healthy volunteers. Our findings confirm that poor metabolizers are underpresented among patients with lung cancer because of a different genetic background. Our findings also reveal that the rare CYP2D6(C) mutant allele is sixfold more frequent among patients with lung cancer (p < 0.0005). This suggests that the CYP2D6(C) allele could be considered as an additional risk factor because carriers could have higher susceptibility to the development of lung cancer.

Immunohistochemical detection of pulmonary cytochrome P450IA and metabolic activities associated with P450IA1 and P450IA2 isozymes in lung cancer patients.

The main polycyclic aromatic hydrocarbon-inducible cytochrome P450 was studied in lung tissue from 57 lung cancer patients by immunohistochemistry, using a monoclonal antibody (1-7-1) that recognizes P450IA1 and P450IA2 isozymes. The intensity of immunostaining was compared with the pulmonary activity of a P450IA1-dependent enzyme, aryl hydrocarbon hydroxylase (AHH), and with P450IA2-related metabolic activity estimated from the ratio of caffeine metabolites in urine. Immunostaining was not observed in peripheral lung tissue of nonsmokers or ex-smokers but was seen in the bronchiolar and alveolar epithelium of all patients who were smokers and had a peripheral carcinoma (16/16) and of 60% (10/17) of those who had a bronchial carcinoma. AHH activity was positively related to the intensity of immunostaining, and an almost 2-fold increase due to smoking was detected in the ratios of caffeine metabolites. These results demonstrate that tobacco smoke induces P450IA1 in the lung and probably P450IA2 in the liver, and suggest a role for certain metabolic phenotypes of P450IA1 in peripheral pulmonary carcinoma.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in bronchial squamous preinvasive lesions.

Metalloproteinases and their inhibitors are known to play an important role in the extracellular matrix remodeling associated with preinvasive lesions and invasive carcinomas; however, little is known about their role in early lung carcinoma. Immunohistochemical studies were made of the reactivity of bronchial squamous preneoplastic lesions from cigarette smokers, including basal cell hyperplasia, squamous metaplasia, dysplasia, carcinoma in situ, and invasive squamous cell carcinoma for matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and type IV collagen in 13 patients. Staining for type IV collagen disclosed discontinuities in basement membranes from basal cell hyperplasia to dysplasia, progressing to destruction in carcinoma in situ and invasive carcinoma. Reactivity for MMP-9 was mild in basal cell hyperplasia and squamous metaplasia, increasing in carcinoma in situ and invasive carcinoma. In contrast, reactivity for MMP-1 was strong in basal cell hyperplasia and squamous metaplasia, decreasing in carcinoma in situ and invasive carcinoma. Some neoplastic cells in carcinoma in situ and invasive carcinoma were MMP-3 positive. Staining for MMP-2 and TIMP-1 was moderate to strong in all squamous preinvasive lesions. Confocal microscopy showed MMP-9-positive cells passing through fragmented basement membranes in which type IV collagen and MMP-9 were colocalized. Type IV collagen colocalized with MMP-2 in all lesions and with TIMP-1 in basal cell hyperplasia and squamous metaplasia. The inverse relationships between the reactivity for MMP-1 and MMP-9 with progression of bronchial squamous preinvasive lesions suggest important roles for these MMPs in basement membrane remodeling in these lesions.

Human alveolar macrophage proteolytic enzyme activities in chronic obstructive pulmonary disease. Lack of correlation with functional abnormalities.

The occurrence of emphysema in people deficient in alpha1-antitrypsin and the production of emphysema in experimental animals with elastolytic enzymes suggest proteolysis as a mechanism for the development of emphysema. To investigate the possible role of pulmonary alveolar macrophages in the pathogenesis of emphysema, we measured elastase, acid protease, and elastase-like esterase activities in macrophages from patients with chronic obstructive lung disease and attempted to correlate the level of enzyme activity with the severity of pulmonary function abnormality measured in these patients. Compared to values for cigarette smokers with normal pulmonary function, these macrophage enzyme activities were not increased in patients with chronic obstructive lung disease, and there was no correlation of high elastase activity with more severe degrees of pulmonary function abnormality. These findings lead us to believe that the absolute level of proteolytic enzymes in pulmonary alveolar macrophages is not in itself a determinant of emphysema.

Long-term survival and toxicity in small cell lung cancer. Expanded Southwest Oncology Group experience.

The Southwest Oncology Group formed a database of 2,501 patients consecutively enrolled in small cell lung cancer (SCLC) trials since 1976. This report summarizes an analysis of this database to determine predictors of 2- and 5-year survival in limited stage disease (LD) and 1- and 2-year survival in extensive stage disease (ED). In addition, we analyzed the frequency of late recurrences, toxicity, and quality of life issues in the long-term survivors. For consecutive studies, greater than or equal to 2-year survival in LD were 15 percent, 21 percent, 28 percent, and 43 percent; 5-year survivals were 5 percent, 9 percent, 8 percent, and 20 percent. In ED, greater than or equal to 1-year survivals were 27 percent, 23 percent, 21 percent, and 42 percent; greater than or equal to 2-year survivals were 6 percent, 5 percent, 3 percent, and 19 percent. Using the logistic regression multivariate model, independent favorable predictors of 5-year survival for patients accrued to our older LD trials were normal lactate dehydrogenase (LDH) values and good performance status. Therapy as employed in these trials was not an independent factor. However, if patients enrolled on more recent trials were included, 2-year predictors could be assessed. Therapy with concurrent chemoradiotherapy and female gender then became additional independent favorable predictors. In ED, a single metastatic site and a normal LHH value were favorable predictors of survival beyond 1 year. The retrospective review of 63 patients with LD who survived at least 5 years found 33 asymptomatic patients with no recurrent disease; 6 with recurrent SCLC, 3 of whom died; 7 who died of non-cancer-related causes or unknown causes; 3 who died of secondary primary lung cancer; and 14 alive with persistent central nervous system symptoms and signs, possibly due to prophylactic brain radiation as given in the first 3 trials. No increased incidence of this syndrome has yet been observed in subsequent trials. For ED patients, 25 of 51 survivors greater than or equal to 2 years subsequently died of recurrent SCLC. The majority of the long-term survivors with ED (35 of 51) had either a single metastatic site or metastases limited to opposite side of the chest or regional nodes. Our multivariate models support the conclusion that aggressive combined modality, concurrent induction therapy, along with favorable prognostic variables, independently contribute to the improved long-term survival we have observed in LD. Longer follow-up is required to confirm that this improvement has occurred with less late toxicity.

Diagnostic yield of adenosine deaminase in bronchoalveolar lavage.

Adenosine deaminase (ADA) activity rises in various body fluids in patients with tuberculosis. A prospective study was conducted to determine the diagnostic value of ADA activity in bronchoalveolar lavage. Between March 2001 and February 2003, 148 patients were enrolled in our study, mean age 55.6 years (SD 14.6), and a male to female ratio of 2.4:1. The mean duration of symptoms was 66.2 days. All patients were either sputum-smear negative for AFB or failed to produce sputum. The final diagnosis resulted in three patient groups: 43 with pulmonary tuberculosis, 70 malignancy, and 35 miscellaneous causes. The mean ADA activity in the bronchoalveolar lavage for the pulmonary tuberculosis, malignancy, and miscellaneous causes groups was 8.98 (95% CI, 3.79-14.17), 7.63 (95% CI, 4.12-11.14), and 11.61 U/l (95% CI, 3.59-19.62), respectively. No difference was detected in the ADA level in the pulmonary tuberculosis vs other groups (p=0.56, one-way ANOVA). A high level of ADA activity was found in non-tuberculous conditions such as bronchogenic carcinoma, pulmonary hemosiderosis, chronic pneumonia with empyema thoracis and chronic myeloid leukemia. We concluded that ADA activity in the bronchoalveolar lavage was not clearly diagnostic of smear-negative pulmonary tuberculosis. Early diagnosis required histopathology of biopsied transbronchial specimens obtained by fiberoptic bronchoscopy.

Status of lipid peroxidation and antioxidant enzymes in malignant (bronchogenic carcinoma) and non-malignant pleural effusions.

Eighty patients from Chennai Medical College (patients with bronchogenic carcinoma) and from Tambaram Tuberculosis Hospital (patients with non-malignant pulmonary diseases mainly tuberculosis) in whom the etiologic diagnosis of their pleural effusions are confirmed were included in the study. Lipid peroxidation (LPO) and activities of antioxidant enzymes were estimated in pleural exudates of the two groups. Lipid peroxidation was found to be increased and the status of antioxidants were found to be decreased in lung malignant pleural exudates when compared to those of non-malignant effusions. The possible reasons for the observed results discussed.

Gastric metastasis and ectopic amylase production in bronchogenic carcinoma.

A case of small cell anaplastic carcinoma of bronchus is described. Primary tumour had metastasized in the stomach and there was production of amylase by the tumour evidenced by raised serum amylase and pleural fluid amylase.

The association of adenosine deaminase activity with T-lymphocytes and subsets in pulmonary tuberculosis and bronchogenic carcinoma.

Simultaneous determination of blood/lung ADA activity and T-lymphocyte subsets was conducted in 12 patients with active pulmonary tuberculosis, 12 patients with bronchogenic carcinoma and 11 healthy volunteers. Differences were significant only in the tuberculosis patients, namely, increased mean enzyme values in both the peripheral blood (36.68 +/- 10.90 U/L) and in the BALF (4.25 +/- 2.19 U/L), and correlation of ADA activity between the blood and the diseased lung only; the difference in elevated enzymatic activity between the tuberculosis group and the cancer group was of no statistical significance. We conclude that simultaneous ADA analysis of the blood and the BALF may be of diagnostic value in cases suspected of having tuberculosis as yet undiagnosed by other means. Based on the lowest value of enzymatic activity in the blood of patients with tuberculosis (28 U/L), the test has a sensitivity of 75 per cent and a specificity of 100 per cent; whereas the lowest value in the BALF of tuberculosis patients (2.9 U/L), the test has a sensitivity of 77 per cent and a specificity of 82 per cent. Findings that there was a blood-lung correlation of elevated ADA activity and a correlation of enzymatic elevation with increased numbers of T-cells bearing IL-2 receptor in cases of pulmonary tuberculosis only provide evidence in support of T-lymphocytes actively participating in the ongoing immune process.

Can't lung cancer patients detoxify procarcinogens?

BACKGROUND: Glutathione S-transferase mu (GST mu) enzyme detoxifies carcinogens in tobacco smoke. We assessed the clinical usefulness of serum assay of GSTm in determining the risk for lung cancer. MATERIALS AND METHODS: Fifty-nine patients with primary lung cancer and 32 control cases were enrolled. GSTm detection was performed by the method ELISA. RESULTS: GSTm enzyme positivity rate of the patient group (39%) was significantly lower than the control group (59.4%) (p < 0.05). The GSTm positivity rates were 28.6% for the non-smoker patients with a cancer history of relatives, 31.6% for the smoker patients with the cancer history of relatives, 14.6% for the non-smoker patients with the lung cancer history of relatives and 16.7% for the smoker patients with the lung cancer history of relatives. CONCLUSIONS: We concluded that if the people lacking GSTm are smokers and have a cancer and/or lung cancer history among their relatives, they would challenge a greater risk of lung cancer than the individuals having GST mu isoenzyme.

[Determination of benzopyrene-hydroxylase (cytochrome P-450) activity in patients with lung cancer]. / Opredelenie aktivnosti fermenta benzpiren-gidroksilazy (tsitokhrom P-450) u bol'nykh rakom legkogo.

The study was concerned with comparison of inducibility index (ii) for benzopyrene-hydroxylase in mitogen-activated lymphocytes in patients with primary cancer of the lung, other cancers (breast, stomach, lower lip, rectum, skin and thyroid gland) and in lymphocytes of blood donors. The highest ii values were registered in bronchogenic carcinoma patients. Smokers, both lung cancer patients and healthy donors, revealed high values (80.0 and 83.3%, respectively). A modified "lymphocytic test" to assess the risk factor for primary lung cancer is suggested.

Cytochrome P450-isoenzyme 1A1 in susceptibility to tobacco-related lung cancer.

BACKGROUND: Tobacco smoke contains many carcinogens that may mediate susceptibility to lung cancer. Cytochrome P450 isoenzyme 1A1 activity and expression increases several fold in lung cancer due to smoking. Finding the role of cytochrome P450 1A1 in susceptibility to tobacco-related lung cancer may be important to predict the outcome in early stage cancer, and may result in an improved survival rate. PATIENTS AND METHODS: This study was carried on 2 groups of patients: group A was 20 patients with operable smoking-related lung cancer, who underwent surgery at the time of diagnosis; group B was 20 nonsmokers without lung cancer who underwent chest exploration following road traffic accidents. Specimens were obtained from tumor tissue and surrounding healthy tissue in group A patients, and from healthy lung tissue in group B patients. These specimens were sent for measurement of protein content and cytochrome P450 1A1 activity. RESULTS: There was significantly greater tissue cytochrome P450 1A1 activity in group A compared to group B. Patients with stage II cancer showed significantly higher levels of tissue cytochrome P450 1A1 activity than those with stage I. There was also a significant difference in tissue cytochrome P450 1A1 activity between the tumor tissue and the tissue surrounding the tumor. CONCLUSION: Carcinogens in smoke increase cytochrome P450 1A1 activity, which might be considered to play a role in cigarette smoking-induced lung cancer.