Bone morphogenetic protein 2 stimulation of tumor growth involves the activation of Smad-1/5.

Morphogenetic protein 2 (BMP-2) is normally expressed in the embryo promoting the development of several organs. Aberrant expression of BMP-2 occurs in approximately 98% of lung carcinomas, however, its role in regulating tumor growth is poorly understood. We show that BMP-2 induces Id-1 expression in lung cancer cell lines through its activation of Smad-1/5, which is dependent on cell culture conditions. A549 cells in DMEM/5% FCS BMP-2 activated Smad-1/5 and caused a transient increase in proliferation. In serum-free medium, BMP-2 induced significantly less Smad-1/5 activation and Id-1 expression, and produced significant growth inhibition. The affect of BMP-2 on tumor growth in vivo was substantially more significant. Recombinant BMP-2 coinjected with A549 cells, into nude mice increased proliferation and produced an increase in Id-1 expression. Forced expression of BMP-2 in A549 cells significantly enhanced tumor growth in the lungs following intravenous injection but not of subcutaneous tumors. Tumors in the lung were found to have an activated Smad-1/5 and expressed Id-1. Subcutaneous tumors expressed less activated Smad-1/5 and Id-1 than that of controls. Human lung carcinomas were also found to express an activated Smad-1/5 and Id-1. We provide evidence that BMP-2 promotes tumor growth. This paper highlights that cell culture experiments may not reveal the full biological affects of BMP-2, and its activity varies depending of the local environment.

High telomerase activity in primary lung cancers: association with increased cell proliferation rates and advanced pathologic stage.

BACKGROUND: Telomerase enzyme activity is not detected in most normal cells, a phenomenon believed to be associated with limitations on cellular proliferation. Since this activity is detected in nearly all human tumors, including non-small-cell lung cancers, it has been suggested that telomerase activation may be coupled to acquisition of the malignant phenotype. In this study, we determined whether telomerase activity was associated with tumor pathologic stage, tumor cell proliferation rates, and clinical outcome in a cohort of patients with resected non-small-cell lung cancer for whom long-term follow-up was available. METHODS: Primary tumor specimens from 99 patients treated with surgery alone and six patients treated with surgery after chemotherapy were analyzed. Telomerase activity was measured by means of a modified Telomeric Repeat Amplification Protocol (TRAP) assay. Southern blot analysis of terminal restriction fragments was used to evaluate telomere length. Immunohistochemical analysis of Ki-67, a proliferation-associated nuclear antigen, was used to assess tumor cell proliferation. RESULTS: Telomerase activity was detected in 84 of the 99 tumors treated with surgery alone; this activity was not detected in specimens of adjacent, benign lung tissue. Telomerase was detected in only three of six tumors resected after chemotherapy. For the surgery-alone group, statistically significant positive associations were found between the level of telomerase activity and tumor stage, lymph node metastasis, pathologic TNM (tumor-node-metastasis) stage, and Ki-67 immunostaining; a statistically significant inverse association was found between telomerase activity and patient age. No statistically significant differences in telomere length were found in relation telomerase activity or pathologic stage. Telomerase activity was not found to be associated with clinical outcome in a multivariate Cox proportional hazards analysis adjusted for tumor stage and lymph node status. CONCLUSIONS: High telomerase activity is detected frequently in primary non-small-cell lung cancers that exhibit high tumor cell proliferation rates and advanced pathologic stage.

Cyclin D1 proteolysis: a retinoid chemoprevention signal in normal, immortalized, and transformed human bronchial epithelial cells.

BACKGROUND: Retinoids (derivatives of vitamin A) are reported to reduce the occurrence of some second primary cancers, including aerodigestive tract tumors. In contrast, beta-carotene does not reduce the occurrence of primary aerodigestive tract cancers. Mechanisms explaining these effective retinoid and ineffective carotenoid chemoprevention results are poorly defined. Recently, the all-trans-retinoic acid (RA)-induced proteolysis of cyclin D1 that leads to the arrest of cells in G1 phase of the cell cycle was described in human bronchial epithelial cells and is a promising candidate for such a mechanism. In this study, we have investigated this proteolysis as a common signal used by carotenoids or receptor-selective and receptor-nonselective retinoids. METHODS: We treated cultured normal human bronchial epithelial cells, immortalized human bronchial epithelial cells (BEAS-2B), and transformed human bronchial epithelial cells (BEAS-2BNNK) with receptor-selective or receptor-nonselective retinoids or with carotenoids and studied the effects on cell proliferation by means of tritiated thymidine incorporation and on cyclin D1 expression by means of immunoblot analysis. We also examined whether calpain inhibitor I, an inhibitor of the 26S proteasome degradation pathway, affected the decline (i.e., proteolysis) of cyclin D1. RESULTS: Receptor-nonselective retinoids were superior to the carotenoids studied in mediating the decline in cyclin D1 expression and in suppressing the growth of bronchial epithelial cells. Retinoids that activated retinoic acid receptor beta or retinoid X receptor pathways preferentially led to a decrease in the amount of cyclin D1 protein and a corresponding decline in growth. The retinoid-mediated degradation of cyclin D1 was blocked by cotreatment with calpain inhibitor I. CONCLUSIONS: Retinoid-dependent cyclin D1 proteolysis is a common chemoprevention signal in normal and neoplastic human bronchial epithelial cells. In contrast, carotenoids did not affect cyclin D1 expression. Thus, the degradation of cyclin D1 is a candidate intermediate marker for effective retinoid-mediated cancer chemoprevention in the aerodigestive tract.

Overexpression of cyclins D1 and E is frequent in bronchial preneoplasia and precedes squamous cell carcinoma development.

Increased protein expression of the G1 cyclins D1 and E is reported in invasive non-small cell lung carcinoma. However, during transformation of the bronchial epithelium, overexpression of these species occurs, and their relationship to aberrant expression of p53 and retinoblastoma (Rb) has not been described previously. To determine the expression of these cell cycle regulators during the development of invasive squamous cell carcinoma (SCC) of the lung, the immunohistochemical expression patterns in normal bronchial epithelium (n = 36), squamous metaplasia (SM; n = 28), and epithelial atypia (n = 34) were compared with that in low-grade dysplasia (LGD; n = 17), high-grade bronchial dysplasia (HGD; n = 30), and SCC (n = 36). Monoclonal anti-p53 Pab1801, polyclonal anti-cyclin D1 DCS6, monoclonal anti-cyclin E HE12, and monoclonal anti-Rb OP-66 antibodies were used. Cyclin D1 was not expressed in normal bronchial epithelium but was detected in 7% of SMs, 15% of atypias; 18% of LGDs, 47% of HGDs, and 42% of SCCs. Cyclin E was not detected in normal epithelium (n = 24), SM (n = 16), or LGD (n = 12), but it was found in 9% of atypias (2 of 22), 33% of HGDs (7 of 21), and 54% of SCCs (13 of 24). p53 was not expressed in normal epithelium, SM, and LGD, but it was overexpressed in 6% of atypias, 53% of HGDs, and 61% of SCCs. Abnormal Rb expression was found only in 2 of 36 cases of SCC. A total of 91% of HGDs and 92% of SCCs exhibited overexpression of at least one of the p53, cyclin D1, or cyclin E species. However, no link was observed between overexpression of p53 and the overexpressed G1 cyclins in preneoplastic lesions. Overexpression of cyclin D1, cyclin E, and p53 occurs frequently and independently in pulmonary SCC and is detected in lesions before the development of invasive carcinoma. In contrast, altered Rb expression is a late and infrequent event in squamous cell carcinogenesis.

Growth suppression of transformed human bronchial epithelial cells by all-trans-retinoic acid occurs through specific retinoid receptors.

The retinoids are reported to chemoprevent second aerodigestive tract cancers in patients with prior lung or head and neck cancers. Since those retinoids already examined in clinical trials do not induce major clinical responses in lung cancers, it is hypothesized that the beneficial chemoprevention activity in lung neoplasias occurs within 'fields' of carcinogen-transformed epithelial cells. To begin to investigate this retinoid action during lung carcinogenesis, the BZR-T33 ras transformed human bronchial epithelial cell line that grows in an anchorage independent manner was examined. This study reports, as compared to controls, that all-trans-retinoic acid (RA)-treatment suppresses BZR-T33 proliferation in monolayer cultures and in anchorage independent and cloning efficiency growth assays. RA induces RAR-gamma 2 > RAR gamma 1 in BZR-T33 cells but expression at the total cellular RNA level of RAR alpha and RXR alpha is not augmented by RA-treatment. RAR beta mRNA expression is repressed before and after RA-treatment and is only detected using a reverse transcription polymerase chain reaction (RT-PCR) assay. To determine directly which of these expressed retinoid receptors signals growth suppression, each receptor was individually transfected into BZR-T33 cells using episomal vector in colony efficiency assays. RAR gamma over-expression in the presence or absence of RA-treatment did not suppress BZR-T33 growth more than controls. In contrast, over-expression of the other examined retinoid receptors inhibited BZR-T33 cellular cloning efficiency prior to RA-treatment and in this decreasing order after RA-treatment: RAR alpha > RAR beta > RXR alpha. The findings reported here reveal that RA suppresses proliferation and cloning efficiency in this transformed human bronchial epithelial cell through specific retinoid receptors. Further work is needed to evaluate the role of RA or its nuclear receptors in inhibiting even earlier steps in lung carcinogenesis.

Inhibited transformation of immortalized human bronchial epithelial cells by retinoic acid is linked to cyclin E down-regulation.

The retinoids are reported to reduce second primary aerodigestive tract tumors in patients with prior lung or head and neck carcinomas. Yet, the optimal retinoid useful for chemoprevention and those mechanisms linked to this chemoprevention are not identified. This study reports an in vitro model for carcinogen-induced transformation of immortalized human bronchial epithelial (BEAS-2B) cells that was adapted to study the anti-carcinogenic effects of all-trans-retinoic acid (RA). Following exposure to carcinogens: cigarette smoke condensate (CSC) or N-nitrosamine-4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK), BEAS-2B cells exhibited evidence of transformation. This included an increased anchorage independent growth or acquired ability to form tumors in athymic mice. This transformation was inhibited by RA as demonstrated by a lack of augmented anchorage independent growth or tumor formation in athymic mice for the cells treated with RA. The BEAS-2B cells transformed by NNK exhibited an increase in cyclin E expression which was associated with an increase in the cyclin E-Cdk2 kinase activity. Over-expression of human cyclin E by transfection shows cyclin E enhances the basal clonal growth of BEAS-2B cells. In both the parental and transformed BEAS-2B cells, RA down-regulated cyclin E protein levels which was associated with an inhibition of growth and an accumulation of cells in G1. The data reported here suggest the decline of cyclin E expression represents a potential mechanism for the RA-induced growth suppression which is linked to the anti-carcinogenic effects of RA. Thus, this study reports the adaption of an in vitro model of lung carcinogenesis suitable to test the activity of chemoprevention agents.

Overexpression of the epidermal growth factor receptor and its ligand transforming growth factor alpha is frequent in resectable non-small cell lung cancer but does not predict tumor progression.

The epidermal growth factor receptor (EGFR) and its ligand transforming growth factor (TGF) alpha are hypothesized to form an autocrine growth loop in non-small cell lung cancer (NSCLC) and to play an important role in tumor formation and progression. We studied the association between overexpression of EGFR, TGF-alpha, or both, and overall survival of patients with resectable NSCLC. Overexpression, defined as >20% of tumor cells staining on immunohistochemistry, was examined in 96 tumor samples from consecutive patients having resection of previously untreated, well-staged NSCLC who were then followed prospectively (median follow-up, 20.7 months). The expression of three other ligands for EGFR (epidermal growth factor, cripto, and amphiregulin) was examined by Northern analysis to determine whether they might also contribute to a potential growth stimulatory loop. Overall, survival was calculated by the method of Kaplan and Meier, and prognostic factors were compared using the log-rank test. Overexpression of EGFR only was found in 32% (31 of 96), of TGF-alpha only in 10% (10 of 96), of both EGFR and TGF-alpha in 38% (37 of 96), and of neither in 19% (19 of 96) of tumors. EGFR and TGF-alpha overexpression was observed in all tumor stages and histological types but was most frequent in squamous cell carcinoma. By univariate and multivariate analyses, only tumor stage, not histology or overexpression of EGFR, TGF-alpha, or both, had a significant impact on overall survival. No expression of epidermal growth factor or cripto was observed at the total cellular RNA level of Northern analysis in tumor or benign lung, suggesting that in NSCLC these ligands may not participate in an autocrine growth stimulatory loop with EGFR. Differential overexpression of amphiregulin in malignant versus normal lung was observed, but this expression pattern did not have a prognostic impact. Thus, EGFR and TGF-alpha overexpression is frequent in early-stage NSCLC but is not associated with a survival difference. These findings suggest that this growth factor/receptor loop is more important for lung tumor formation than for tumor progression.

Red cell deformability is an early indicator of infection.

Red blood cells (RBC) have been shown to become less deformable during infection. The RBC deformability index (DI) was measured within 24 hours of admission in 37 patients who had suffered trauma and every 48 to 72 hours thereafter while they were in the surgical intensive care unit to assess whether DI could be used as an early indicator of infection after injury. Infection was defined as a temperature of 101 degrees F or more and a white blood cell count of more than 12,000/cm3 associated with a positive culture. Eighteen patients developed an infection, and 19 patients did not. On day 1, both groups showed a significant decrease in DI, compared to controls (0.33 +/- 0.18 and 0.34 +/- 0.25 for patients with infection and patients with no infection vs 1.52 +/- 0.12 for control volunteers; p less than 0.05). In the group with no infection, the DI improved in 16 of 19 patients after injury; the DI in patients with infection continued to decrease in 17 of 18 patients. The decrease in DI occurred 4 +/- 2 days (range, 2 to 8 days) before the diagnosis of infection. No significant differences were apparent in the absolute white blood cell count between the group with infection and the group with no infection at any time after injury. Differences in maximal temperature were noted on day 3 and beyond; however, 30% of patients with no infection had a temperature of more than 101 degrees F for 7 days. These data show that trauma results in a significant decrease in RBC deformability and that serial changes in DI appeared to predict which patients would develop an infection and which patients would recover uneventfully. RBC deformability may be helpful in early detection of infection in patients who have suffered trauma.

Correlation between red blood cell deformability and changes in hemodynamic function.

BACKGROUND: In sepsis red blood cells (RBCs) have been shown to be less deformable (i.e., more rigid) and have been implicated in decreasing nutrient blood supply and possibly leading to organ dysfunction. However, no studies have demonstrated an association between organ dysfunction and rigid RBCs. This study examined cardiovascular physiologic and histologic changes in two different models to determine whether a relationship may exist between RBC deformability and organ function. METHODS: In the following two experiments, cardiac index (CI) was continuously measured, whereas both deformability index and histology were examined at the end of the experimental periods. The first experiment studied nonanesthetized, hydrated rats after a cecal ligation and puncture (CLP), a slow-developing means of inducing RBC rigidity. In a second experiment animals were anesthetized and received a 20% total blood volume transfusion of either diamide-treated (rigid) RBCs or normal RBCs. RESULTS: CLP-treated animals' CI gradually decreased during 18 hours (232 +/- 60 ml/min/kg to 123 +/- 90 ml/min/kg; p = 0.05), with an increase in systemic vascular resistance (1459 +/- 517 dyne.sec/cm5.m2 to 2337 +/- 1213 dyne.sec/cm5.m2; p = 0.02). Diamide-treated animals had a rapid decrease in CI (86 +/- 7.0 ml/min/kg to 58 +/- 13 ml/min/kg; p = 0.05) and increase in SVR (2269 +/- 373 dyne.sec/cm5.m2 to 3897 +/- 988 dyne.sec/cm5.m2; p = 0.05) from baseline to 120 minutes after treatment respectively. The DI was significantly lower in both CLP and diamide groups (p < 0.03) when compared with control animals. Histologic evidence of subendocardial necrosis was shown in both the CLP- and Diamide-treated animals. CONCLUSIONS: These data suggest an association with RBC deformability and organ function in both septic and nonseptic animal models.

Current management of thymoma.

Patients with thymoma present rarely even on active thoracic surgery services. These patients may suffer from many associated conditions but the most common is myasthenia gravis. Aggressive surgical resection is the mainstay of initial therapy. Radiation therapy has a role in patients who are left with retained neoplasm after surgical resection. Recurrence may occur at prolonged intervals but should be treated aggressively.

Economic analysis in health care antitrust.

The Federal Trade Commission (FTC or Commission) has been very active in enforcing antitrust laws in the health care field for the past two decades. The staff has investigated a wide variety of cases covering a broad range of restrictions on competition. These cases can be divided into three basic types of cases in health care: (1) mergers and acquisitions, (2) horizontal restraints cases or agreements among competitors, and (3) input market monopolization cases, such as hospital privileges cases. The Commission relies on both legal and economic analysis in all of these cases. As Chairman Steiger of the Federal Trade Commission has stated, antitrust policy has been "increasingly reshaped by analysis based on economic theory." This article attempts to explain the economic analysis used in antitrust enforcement as applied to the first two of the three types of health care cases. Section I presents the basic economic framework that is used to assess the competitive implications of health care mergers and acquisitions. Section II describes the analysis applied to other agreements among competitors in the health care field and briefly explains how this analysis differs in other health care cases.

Quantitative analysis of fuel-related hydrocarbons in surface water and wastewater samples by solid-phase microextraction.

Solid-phase microextraction (SPME) parameters were examined on water contaminated with hydrocarbons including benzene and alkylbenzenes, n-alkanes, and polycyclic aromatic hydrocarbons (PAHs). Absorption equilibration times ranged from several minutes for low molecular weight compounds such as benzene to 5 h for high molecular weight compounds such as benzo[a]pyrene. Under equilibrium conditions, SPME analysis with GC/FID was linear over 3-6 orders of magnitude, with linear correlation coefficients (r(2)) greater than 0.96. Experimentally determined FID detection limits ranged from ∼30 ppt (w/w hydrocarbon/sample water) for high molecular weight PAHs (e.g., MW > 202) to ∼1 ppb for low molecular weight aromatic hydrocarbons. Experimental distribution constants (K) were different with 100- and 7-µm poly(dimethylsiloxane) fibers, and poor correlations with previously published values suggest that K depends on the fiber coating thickness and the sorbent preparation method. The sensitivity of SPME analysis is not significantly enhanced by larger sample volumes, since increasing the water volume (e.g., from 1 to 100 mL) has little effect on the number of analyte molecules absorbed by the fiber, especially for compounds with K < 500. Water sample storage should utilize silanized glassware, since hydrocarbon losses up to 70% could be attributed to unsilanized glassware walls when samples were stored for 48 h. Hydrocarbon losses at part-per-billion concentrations also occurred with surface waters due to partitioning onto part-per-thousand concentrations of suspended solids. Quantitative determinations of aromatic and aliphatic hydrocarbons (e.g., in gasoline-contaminated water) can be performed using GC/MS with deuterated internal standard or standard addition calibration as long as the target components or standards had unique ions for quantitation or sufficient chromatographic resolution from interferences. SPME analysis gave good quantitative performance with surface waters having high suspended sediment contents, as well as with coal gasification wastewater which contained matrix organics at 10(6)-fold higher concentrations than the target aromatic hydrocarbons. Good agreement was obtained between a 45-min SPME and methylene chloride extraction for the determination of PAH concentrations in creosote-contaminated water, demonstrating that SPME is a useful technique for the rapid determination of hydrocarbons in complex water matrices.

Posttranslational regulation of cyclin D1 by retinoic acid: a chemoprevention mechanism.

The retinoids are reported to reduce incidence of second primary aerodigestive cancers. Mechanisms for this chemoprevention are previously linked to all-trans retinoic acid (RA) signaling growth inhibition at G1 in carcinogen-exposed immortalized human bronchial epithelial cells. This study investigated how RA suppresses human bronchial epithelial cell growth at the G1-S cell cycle transition. RA signaled growth suppression of human bronchial epithelial cells and a decline in cyclin D1 protein but not mRNA expression. Exogenous cyclin D1 protein also declined after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms were active in this regulation of cyclin D1 expression. Findings were extended by showing treatment with ubiquitin-dependent proteasome inhibitors: calpain inhibitor I and lactacystin each prevented this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, did not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appeared after proteasome inhibitor treatments, suggesting that ubiquitinated species were present. To learn whether RA directly promoted degradation of cyclin D1 protein, studies using human bronchial epithelial cell protein extracts and in vitro-translated cyclin D1 were performed. In vitro-translated cyclin D1 degraded more rapidly when incubated with extracts from RA treated vs. untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depended on the C-terminal PEST sequence, a region rich in proline (P), glutamate (E), serine (S), and threonine (T). Taken together, these data highlight RA-induced cyclin D1 proteolysis as a mechanism signaling growth inhibition at G1 active in the prevention of human bronchial epithelial cell transformation.