Pulmonary non-tuberculous mycobacteria in a general respiratory population.

The prevalence of non-tuberculous mycobacterium (NTM) appears to be increasing. Much of the experience in the literature about this emerging organism comes from specialised units or populations such as cystic fibrosis patients. We, therefore, aim to evaluate the experience in a general respiratory population of dealing with patients with positive culture of NTM. We did a retrospective review of medical notes of general respiratory patients from whom NTM were isolated from January 2007 to July 2012. Cystic fibrosis patients were excluded. We reviewed 37 patients' (19 males, 18 females) medical records. A total of 73 positive cultures were reviewed. 28 isolates were from sputum samples alone, 34 isolates were from bronchoalveolar lavage alone and 11 isolates were from a combination of sputum and bronchoalveor lavage (11 isolates), We found that Mycobacterium avium was the most frequently isolated Mycobacterium in our laboratory with 22 (60%) patients had Mycobacterium avium in their pulmonary cultures. Interestingly, Mycobacterium gordonae and mycobacterium intracellulare were the second commonest mycobacterium (4, 11%) cultured. We noted 2 (5%), cases of Mycobacterium szulgai, 2 (5%) cases of Mycobacterium chelonae and 2 (5%) cases of Mycobacterium abscessus. There was 1(3%) case of Mycobacterium malmoense. There is prevalence of NTM in male COPD patients (7, 89%) and femal bronchiectasis (10, 77%) patients. Of our 8 COPD patients, 6 (75%) were on inhaled corticosteroids while 2 (25%) were not. 9 (24%) patients were smokers, 11 (30%) were ex-smokers, 14 (38%) were non-smokers and the smoking status of the remaining 3 (8%) was unknown. Of the 37 patients, only 6 (16%) received treatment. However, 2 patients stopped their treatment due to treatment toxicity. We concluded that the isolation of NTM is not uncommon. Defining NTM disease is difficult and deciding which patient to be treated needs careful evaluation as treatment can potentially be very toxic.

COPD exacerbations -- a comparison of Irish data with European data from the ERS COPD Audit.

The European Respiratory Society COPD audit was a cross-sectional, multicentre study that analysed outcomes for COPD patients admitted to hospital with an exacerbation across Europe. We present the data on patients admitted to 11 Irish hospitals that participated in the audit. Among 237 patients (123 Male), the median age was 71 years and 79 (33%) patients were current smokers. 82 (35%) patients received high-flow oxygen before admission and 43 (18%) were cared for in a dedicated respiratory ward. 54 (23%) patients required ventilatory support. Median length of stay was 7 days, 98 (41%) patients were readmitted and 211 (89%) patients were alive at the 90 day follow up point. Irish patients were more likely to receive high-flow oxygen before admission, less likely to be managed in a dedicated respiratory ward and had a higher likelihood of readmission or death within 90 days than the European average.

Overwhelming support among urban Irish COPD patients for lung cancer screening by low-dose CT scan.

PURPOSE: The National Lung Screening Trial (NLST) has renewed interest in low-dose computed tomography (LDCT) screening for lung cancer. Smokers may be less receptive toward LDCT screening, however, compared with never smokers. The views of patients with COPD, a particularly high-risk group, toward LDCT screening for lung cancer are currently unknown. We therefore evaluated attitudes of patients with COPD toward LDCT screening for lung cancer. METHODS: Interviews with Irish patients with COPD who satisfied NLST eligibility criteria were conducted in clinical settings using a questionnaire based on that of a comparable study of U.S. current/former smokers of unspecified disease status. RESULTS: A total of 142 subjects had a mean age of 65.09 ± 6.07 years (46.4 % were male, mean pack years 54.5 ± 33.3, mean FEV1 59.16 ± 23 %); 97.8 % had an identifiable usual source of healthcare. Compared with data from a U.S. cohort of current/former smokers, a higher proportion of Irish COPD smokers: believed that they were at risk for lung cancer (63.6 vs. 15.7 %); believed that early detection improved chances of survival (90 vs. 51.2 ); were willing to consider LDCT screening (97.9 vs. 78.6 %); were willing to pay for a LDCT scan (68.6 vs. 36.2 %); and were willing to accept treatment recommendations arising (95.7 vs. 56.2 %; p < 0.0001 for all comparisons). CONCLUSIONS: Urban Irish smokers with COPD who would be eligible for LDCT screening are almost universally in favor of being screened and treated for screening-detected lung cancers. This readily accessible high-risk population should be actively targeted in future screening programs.

Overexpression of E2F-1 in glioma triggers apoptosis and suppresses tumor growth in vitro and in vivo.

The transfer of apoptosis genes to tumors is one of the most promising strategies for cancer gene therapy. We have shown that massive apoptosis occurs when wild-type p53 expression is induced in glioma cells carrying a p53 gene mutation. However, adenovirus-mediated p53 gene transfer is ineffective in causing apoptosis in glioma cells that retain a wild-type p53 genotype. We evaluated the effect of E2F-1 overexpression on the growth of gliomas in vitro and in vivo. In the in vitro study, the adenovirus-mediated transfer of exogenous E2F-1 protein precipitated generalized apoptosis in gliomas. The treatment with Ad5CMV-E2F-1 of nude mice carrying subcutaneous gliomas arrested tumor growth. Our results indicate that E2F-1 has anti-glioma activity in vitro and in vivo.

Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer.

A retroviral vector containing the wild-type p53 gene under control of a beta-actin promoter was produced to mediate transfer of wild-type p53 into human non-small cell lung cancers by direct injection. Nine patients whose conventional treatments failed were entered into the study. No clinically significant vector-related toxic effects were noted up to five months after treatment. In situ hybridization and DNA polymerase chain reaction showed vector-p53 sequences in posttreatment biopsies. Apoptosis (programmed cell death) was more frequent in posttreatment biopsies than in pretreatment biopsies. Tumor regression was noted in three patients, and tumor growth stabilized in three other patients.

Herbal medicine--sets the heart racing!

The potential for pharmaceuticals to produce side effects and drug interactions is well known to medical practitioners and the lay public alike. However, the potential for alternative medicines to produce such effects is less widely known. We describe a potentially dangerous interaction between a herbal medicine and concomitant selective serotonin re-uptake inhibitor (SSRI) ingestion.

Molecular mediators of cell death in multistep carcinogenesis: a path to targeted therapy.

A consistent, if not invariant, feature of cancer cells is the acquired ability to evade apoptosis. The pioneering work of Dr. Stan Korsmeyer was invaluable in characterizing the molecular foundations of cell death signaling mechanisms during normal development and during multistep carcinogenesis. This foundation now forms the basis for the rational design of therapeutic strategies to selectively activate cell death in cancer cell populations. These strategies are currently being evaluated in an increasing number of clinical trials targeting diverse tumor types.

Deregulated Bcl-2-immunoglobulin transgene expands a resting but responsive immunoglobulin M and D-expressing B-cell population.

We characterized the basis for the follicular lymphoproliferation in transgenic mice bearing a Bcl-2-immunoglobulin (Bcl-2-Ig) minigene representing the t(14;18) of human follicular lymphoma. Discriminatory S1 nuclease protection assays revealed that the Bcl-2-Ig transgene was overexpressed relative to endogenous mouse Bcl-2 in spleen and thymus. Western (immunoblot) analysis demonstrated the overproduction of the human 25-kilodalton Bcl-2 protein, which arose from the transgene, in spleen, thymus, and the expanded B-cell subset. Despite the generalized lymphoid pattern of deregulation, two-color flow cytometry and density gradient centrifugation indicated that the expanded lymphocytes were predominantly small, resting B cells coexpressing B220, immunoglobulin M (IgM), IgD, Ia, and kappa. Cell cycle analysis confirmed that about 97% of these expanded B cells reside in G0/G1. An extensive characterization of transgenic lines revealed a fourfold excess of IgM-IgD-expressing B cells in spleen and dramatically increased numbers in bone marrow. While resting, these cells proliferated in response to lipopolysaccharide and anti-IgM and demonstrated normal B-cell colony formation in soft agar. Moreover, these B cells, which demonstrated an extended survival in vitro even in the absence of stroma, were also resting in G0, yet were capable of proliferative responses. These findings provide consistent evidence that the accumulation of B cells after Bcl-2 overproduction is secondary to prolonged cell survival and not increased cell cycling. This suggests a unique role for Bcl-2 as a proto-oncogene that enhances cell survival independent of promoting cell division.

Characterization of p53 and p21 functional interactions in glioma cells en route to apoptosis.

BACKGROUND: Alterations of the p53 (also called TP53) gene are one of the most common abnormalities in gliomas. We have previously reported that restoration of wild-type p53 protein function in glioma cells results in programmed cell death (apoptosis). Since p53 functions are mediated by genes that directly control the tumor suppressor effect of the p53 protein, understanding the relationship between p53 and p53-related genes in glioma cells will aid in the design of more rational treatment strategies for brain tumors. PURPOSE: We conducted this study to examine the timing of the p53-mediated events preceding apoptosis. More specifically, we undertook this work to characterize the genetic and cell cycle-related factors that may increase the resistance of glioma cells to p53-induced apoptosis. METHODS: Two human glioma cell lines (U-251 MG and U-373 MG) that express mutant p53 protein and two (U-87 MG and EFC-2) that express wild-type p53 protein were used. Replication-deficient adenovirus was utilized as an expression vector to transfer exogenous p53 and p21 complementary DNAs into the glioma cells; control cells were infected with the viral expression vector alone. To monitor gene transfer and the expression of exogenous genes (as well as the expression of endogenous genes), we used western blot analyses and immunohistochemistry analyses. Flow cytometry studies of cellular DNA content were performed to determine the cell cycle phenotype of the glioma cells before and after treatment. RESULTS: p53-mediated apoptosis was preceded by elevation in the levels of the p21 (cell cycle-related) and Bax (apoptosis-related) proteins. In addition, cell cycle analyses showed that glioma cells were arrested in the G2 phase before undergoing cell death. Transfer of p21 induced a G2 block but did not induce apoptosis. Moreover, coexpression of p21 and p53 prevented glioma cells from undergoing apoptosis. Expression of exogenous p53 in wild-type p53 cells did not induce elevation of Bax levels, arrest in G2 phase, or apoptosis. CONCLUSIONS AND IMPLICATIONS: Our data confirmed the ability of wild-type p53 to induce apoptosis in p53 mutant glioma cells. In addition, our results document that p21 plays a role in protecting cells from p53-mediated programmed cell death and suggest that p53-mediated apoptosis and p21 induction may represent, at least in certain cases, opposite signals. Finally, our data suggest that over expression of p21 in gliomas may be related to resistance to treatments that induce apoptosis.

Regulation of the proto-oncogenes bcl-2 and c-myc by the Wilms' tumor suppressor gene WT1.

The Wilms' tumor gene WT1 functions as a tumor suppressor gene, repressing transcription of several growth factors and growth factor receptors. The bcl-2 and c-myc proto-oncogenes are essential for regulation of apoptosis and cell proliferation with roles in development and oncogenesis. We found that WT1 can repress transcription of both the bcl-2 and c-myc promoters. This suggests that WT1 regulates bcl-2 and c-myc during renal development, and the loss of functional WT1 results in deregulation of bcl-2 and c-myc, contributing to tumor formation.

Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer.

The significance of apoptosis in relation to the development and progression of prostate cancer remains largely undefined. bcl-2 is an oncogene that functions by overriding apoptosis. bcl-2 expression was localized to the basal epithelial cells in the normal human prostate with the use of immunohistochemistry. Androgen-dependent and androgen-independent prostate carcinomas were evaluated immunohistochemically for bcl-2 expression. bcl-2 was undetectable in 13 of 19 cases of androgen-dependent cancers. In contrast, androgen-independent cancers displayed diffuse, high levels of bcl-2 staining (P < 0.01). In rats, steady-state levels of bcl-2 mRNA, assessed by S1 assays, reached maximum levels 10 days following castration. Addition of exogenous testosterone with, or without, flutamide demonstrated that the increased bcl-2 mRNA resulted from androgen ablation. Our findings indicate that bcl-2 expression is augmented following androgen ablation and is correlated with the progression of prostate cancer from androgen dependence to androgen independence.

Adenoviral Bak overexpression mediates caspase-dependent tumor killing.

One of the most promising strategies in cancer gene therapy is adenoviral transfer of proapoptotic genes. We therefore evaluated the novel strategy of adenovirally overexpressing the proapoptotic Bak gene. Our results showed marked apoptosis in cancer cells in vivo and in vitro after Bak gene transfer via a binary adenoviral vector system. This effect was not seen in a caspase 3-defective cell line (MCF-7) and was abrogated in Bak-sensitive tumors after administration of the caspase inhibitor z-DEVD-fmk. Our results suggest that adenoviral-mediated overexpression of Bak provides a novel therapeutic strategy for cancer therapy, but this process appears to be caspase dependent.

Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis.

Wild-type p53 is involved in several aspects of cell cycle control and suppression of transformation, inducing either apoptosis or G1 block in cell cycle progression. Using a recombinant adenovirus containing the wild-type p53 cDNA, the biological effects of the newly expressed wild-type p53 protein were examined in six human glioma cell lines. Three cell lines (U-251 MG, U-373 MG, and A-172) expressed endogenous mutant p53, and the other three (U-87 MG, EFC-2, and D54 MG) expressed wild-type p53. The restoration of normal p53-encoded protein in the mutant cell lines induced apoptosis as assessed by morphological studies using nuclear staining, electron microscopy, and flow cytometric assays. In wild-type p53 cell lines, however, the overexpression of wild-type p53 did not result in apoptosis but inhibited cellular proliferation rather drastically and modified the neoplastic phenotype. Differential effects suggest two pathways for glioma oncogenesis and a possible therapeutic strategy.

Apoptosis induction mediated by wild-type p53 adenoviral gene transfer in squamous cell carcinoma of the head and neck.

Cancer gene therapy strategies for inducing apoptosis in solid tumors may allow contemporary medicine to reassess its management of these cancers. We demonstrated previously that overexpression of the wild-type p53 gene in squamous cell carcinoma of the head and neck cell lines via adenovirus-mediated gene transfer suppressed growth both in vitro and in vivo. Here, we characterize the mechanism of the growth suppression by the exogenous p53 gene as a consequence of programmed cell death (apoptosis). One of the cell lines used in this study, Tu-138, harbors a mutated p53 gene, whereas the other cell line, MDA 686LN, possesses a wild-type p53 gene. DNA fragmentation was detected by electrophoresis in both cell lines after infection with the wild-type p53 adenovirus, Ad5CMV-p53. With the use of the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method, 4.4% of the remaining viable Tu-138 cell population was identified as apoptotic as early as 15 h after inoculation with Ad5CMV-p53. The percentage of apoptotic cells increased to 31% at 22 h. In contrast, only 10% of the viable MDA 686LN cells (wt-p53) had undergone apoptosis 30 h after Ad5CMV-p53 infection, although the percentage of apoptotic cells rapidly increased to 60% at 48 h after infection. For in vivo analysis of apoptosis, nude mice in which squamous cell carcinoma of the head and neck cell lines had been implanted s.c. had exogenous wt-p53 transiently introduced to the tumor cells via Ad5CMV-p53 2 days later. In situ end labeling clearly illustrated apoptosis in the tumor cells. These results suggest that wt-p53 plays an important role in the induction of apoptosis in human head and neck cancer cell lines and that selective induction of apoptosis in cancer cells can be further explored as a strategy for cancer gene therapy.

Regulation of Akt/PKB activity, cellular growth, and apoptosis in prostate carcinoma cells by MMAC/PTEN.

Understanding the functional roles of the molecular alterations that are involved in the oncogenesis of prostate cancer, the second most frequent cause of cancer-related deaths among men in the United States is the focus of numerous investigations. To examine the possible significance of alterations associated with the tumor suppressor gene, MMAC/PTEN, in prostate carcinoma, the biological and biochemical effects of MMAC/PTEN expression were examined in LNCaP cells, which are devoid of a functional gene product. Acute expression of MMAC/PTEN via an adenoviral construct resulted in a dose-dependent and specific inhibition of Akt/PKB activation, consistent with the phosphatidylinositol phosphatase activity of MMAC/PTEN. MMAC/PTEN expression induced apoptosis in LNCaP cells, although to a lesser extent than that observed with p53 via an adenoviral construct. However, MMAC/PTEN expression produced a growth inhibition that was significantly greater than that achieved with p53. Overexpression of Bcl-2 in LNCaP cells blocked MMAC/PTEN- and p53-induced apoptosis but not the growth-suppressive effects of MMAC/ PTEN, suggesting that the growth regulatory effects of MMAC/PTEN involve multiple pathways. These studies further implicate the loss of MMAC/PTEN as a significant event in prostate cancer and suggest that reintroduction of MMAC/PTEN into deficient prostate cancer cells may have therapeutic implications.

Transfer of E2F-1 to human glioma cells results in transcriptional up-regulation of Bcl-2.

Strong evidence exists to support the tenet that activation of E2F transcription factors, via alterations in the p16-cyclin D-Rb pathway, is a key event in the malignant progression of most human malignant gliomas. The oncogenic ability of E2F has been related to the E2F-mediated up-regulation of several proteins that positively regulate cell proliferation. However, E2F may indirectly enhance proliferation by activating antiapoptotic molecules. In this work, we sought to ascertain whether E2F-1-mediated events involve the up-regulation of the antiapoptotic molecule Bcl-2. Western blot analyses showed up-regulation of Bcl-2 but not of Bcl-x(L) by 24 h after the transfer of E2F-1. Northern blot studies showed that transfer of E2F-1 also up-regulated Bcl-2 RNA. In support of these findings and the concept that E2F-1 has a direct effect in the induction of Bcl-2, we found a putative E2F binding site within the Bcl-2 sequence. Subsequent gel-mobility shift and supershift experiments involving the CTCCGCGC site in the bcl-2 promoter showed that E2F-1 bound Bcl-2. Transactivation experiments consistently showed that ectopic E2F-1 activated responsive elements located in the -1448/-1441 region in the P1 promoter region of the bcl-2 gene. As expected, other members of the E2F family of transcription factors such as E2F-2 and E2F-4 also transactivated the bcl-2 promoter. Our results demonstrate that E2F-1 modulates the expression of the antiapoptotic molecule Bcl-2 and suggest that up-regulation of Bcl-2 may favor the oncogenic role of E2F-1 and other members of the E2F family of transcription factors.

TRAIL (APO-2L) induces apoptosis in human prostate cancer cells that is inhibitable by Bcl-2.

To determine if TRAIL-induced apoptosis in human prostate tumor cells was suppressed by bcl-2, we compared the levels of apoptosis induced by recombinant human TRAIL in pairs of isogenic cell lines that do or do not express bcl-2. Three human prostate tumor cell lines (PC3, DU145 and LNCaP) and their bcl-2-expressing counterparts were tested for their susceptibility to TRAIL. Cells were exposed to TRAIL in the presence of cycloheximide which acted as a sensitizer. Apoptosis was induced rapidly in PC3 and DU145 neo-control transfected cells, whereas induction in LNCaP required 24 h. All three cell line variants expressing bcl-2 were resistant to the apoptotic effects of TRAIL. Caspase 3 and 8 activation was also detected in the neo control cells after treatment with TRAIL, demonstrating the rapid activation of the caspase cascade similar to that seen with other death receptors. Bcl-2 overexpression in these cells blocked activation of these caspases, suggesting that bcl-2 expression of human cancer cells may be a critical factor in the therapeutic efficacy of TRAIL.

Bcl-2 accelerates multistep prostate carcinogenesis in vivo.

The impact of bcl-2 proto-oncogene expression on the pathogenesis and progression of prostate cancer was examined in a transgenic mouse model. Probasin-bcl-2 transgenic mice were crossed with TRAMP (TRansgenic Adenocarcinoma Mouse Prostate) mice that express the SV40 early genes (T/t antigens) under probasin control. Prostate size, cell proliferation, apoptosis, and the incidence and latency of tumor formation were evaluated. The double transgenic, probasin-bcl-2 X TRAMP F1 (BxT) mice exhibited an increase in the wet weight of the prostate. This was associated with an increase in proliferation, attributable to T/t antigens, and a decrease in apoptosis attributable to bcl-2. The latency to tumor formation was also decreased in the BxT mice compared to the TRAMP mice. The incidence of metastases was identical in both the TRAMP and BxT mice. Lastly, the incidence of hormone-independent prostate cancer was reduced in the BxT mice compared to the TRAMP mice. Together, these results demonstrate that bcl-2 can facilitate multistep prostate carcinogenesis in vivo.

Resistance to radiation-induced apoptosis in Bcl-2-expressing cells is reversed by depleting cellular thiols.

The mechanism by which Bcl-2 oncogene expression inhibits radiation-induced apoptosis has been investigated in two mouse lymphoma cell lines: line LY-as is radiation sensitive, displays substantial radiaton-induced apoptosis, and expresses low levels of Bcl-2; line LY-ar is radiation-resistant, displays a low apoptosis propensity, and expresses 30-fold higher amount of Bcl-2 protein than does the sensitive line. We observed that upon incubation in cystine/methionine-free (C/M-) medium, radiation-induced apoptosis in the LY-ar cells was restored to levels comparable to that seen in the LY-as cells. lntracellular glutathione (GSH) concentrations in LY-ar cells incubated in C/M- medium plummeted to 50% of control values within 2 h. LY-ar cells treated with diethyl maleate (DEM) or diamide, agents that deplete cellular thiols, had increased susceptibility to radiation-induced apoptosis in a manner similar to C/M- medium. These results are consistent with the general idea that Bcl-2 expression blocks apoptosis through an antioxidant pathway that involves cellular thiols. That Bcl-2-expressing tumor cells can be sensitized by exogeneous agents that modify cellular thiols offers strategies for overcoming such resistance.