Unique and shared systemic biomarkers for emphysema in Alpha-1 Antitrypsin deficiency and chronic obstructive pulmonary disease.

BACKGROUND: Alpha-1 Antitrypsin (AAT) deficiency (AATD), the most common genetic cause of emphysema presents with unexplained phenotypic heterogeneity in affected subjects. Our objectives to identify unique and shared AATD plasma biomarkers with chronic obstructive pulmonary disease (COPD) may explain AATD phenotypic heterogeneity. METHODS: The plasma or serum of 5,924 subjects from four AATD and COPD cohorts were analyzed on SomaScan V4.0 platform. Using multivariable linear regression, inverse variance random-effects meta-analysis, and Least Absolute Shrinkage and Selection Operator (LASSO) regression we tested the association between 4,720 individual proteins or combined in a protein score with emphysema measured by 15th percentile lung density (PD15) or diffusion capacity (DLCO) in distinct AATD genotypes (Pi*ZZ, Pi*SZ, Pi*MZ) and non-AATD, PiMM COPD subjects. AAT SOMAmer accuracy for identifying AATD was tested using receiver operating characteristic curve analysis. FINDINGS: In PiZZ AATD subjects, 2 unique proteins were associated with PD15 and 98 proteins with DLCO. Of those, 68 were also associated with DLCO in COPD also and enriched for three cellular component pathways: insulin-like growth factor, lipid droplet, and myosin complex. PiMZ AATD subjects shared similar proteins associated with DLCO as COPD subjects. Our emphysema protein score included 262 SOMAmers and predicted emphysema in AATD and COPD subjects. SOMAmer AAT level <7.99 relative fluorescence unit (RFU) had 100% sensitivity and specificity for identifying Pi*ZZ, but it was lower for other AATD genotypes. INTERPRETATION: Using SomaScan, we identified unique and shared plasma biomarkers between AATD and COPD subjects and generated a protein score that strongly associates with emphysema in COPD and AATD. Furthermore, we discovered unique biomarkers associated with DLCO and emphysema in PiZZ AATD. FUNDING: This work was supported by a grant from the Alpha-1 Foundation to RPB. COPDGene was supported by Award U01 HL089897 and U01 HL089856 from the National Heart, Lung, and Blood Institute. Proteomics for COPDGene was supported by NIH 1R01HL137995. GRADS was supported by Award U01HL112707, U01 HL112695 from the National Heart, Lung, and Blood Institute, and UL1TRR002535 to CCTSI; QUANTUM-1 was supported by the National Heart Lung and Blood Institute, the Office of Rare Diseases through the Rare Lung Disease Clinical Research Network (1 U54 RR019498-01, Trapnell PI), and the Alpha-1 Foundation. COPDGene is also supported by the COPD Foundation through contributions made to an Industry Advisory Board that has included AstraZeneca, Bayer Pharmaceuticals, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Novartis, Pfizer, and Sunovion.

Navigation programs, are they helpful for perioperative care with thyroid cancer patients?

The purpose of this study was to develop and evaluate a navigation program for patients with thyroid cancer. The navigation program was developed following an analysis of the unmet needs of patients who underwent surgery for thyroid cancer. Ninety-nine patients in the control group received usual care, and 95 in the navigation group were managed with a navigation program during the perioperative period. The effectiveness of the navigation program was assessed by administering a questionnaire to both groups. Overall satisfaction scores were significantly higher in the navigation than in the control group (p = .025), as were satisfaction scores on the continuity of information (p < .001), the continuity of management (p = .002), the continuity of relationships with healthcare providers (p<.001), and patient empowerment (p < .001). The newly developed navigation program for patients with thyroid cancer was effective in raising satisfaction levels and in actively managing the disease during the perioperative period.

Aerosol delivery of eukaryotic translation initiation factor 4E-binding protein 1 effectively suppresses lung tumorigenesis in K-rasLA1 mice.

Conventional radiotherapy or chemotherapy for the long-term survival of patients with lung cancer is still difficult for treatment in metastatic and advanced tumors. Therefore, the safe and effective approaches to the treatment of lung cancer are needed. In this study, the effect of delivered eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) on lung cancer progression was evaluated. Recombinant adeno-associated virus (rAAV)-M3/4E-BP1 was delivered into 6-week-old K-rasLA1 lung cancer model mice through a nose-only inhalation system twice a week for 4 weeks. Long-term repeated delivery of 4E-BP1 effectively reduced tumor progression in the lungs of K-rasLA1 mice. Reduction of eIF4E by overexpression of 4E-BP1 resulted in suppression of cap-dependent protein expression of basic fibroblast growth factor (bFGF or FGF-2) and vascular endothelial growth factor (VEGF). In addition, delivered 4E-BP1 inhibited the proliferation of lung cancer cells in K-rasLA1 mice model. Our results suggest that long-term repeated viral delivery of 4E-BP1 may provide a useful tool for designing lung cancer treatment.

Co-delivery of LETM1 and CTMP synergistically inhibits tumor growth in H-ras12V liver cancer model mice.

As hepatocellular carcinoma (HCC) is one of the most common tumors worldwide, development of novel therapeutic approaches for HCC is urgently needed. Two different genes, LETM1 and CTMP, which target mitochondrial functions, were chosen and linked using 2A-peptide sequence. Successful self-cleavage of 2A-peptide induced synergistic antitumor effect in the liver of H-ras12V, the HCC model mice, by simultaneous activation of LETM1 (Leucine zipper/EF hand-containing transmembrane-1) and CTMP (carboxyl-terminal modulator protein). Overexpression of LETM1 and CTMP significantly reduced the incidence of tumorigenesis, which were confirmed by gross and microscopic observations. Morphological changes in mitochondria, such as swelling and loss of cristae, were significant, and the prolonged activation of defects in mitochondrial function led to mitochondria-mediated apoptosis. Furthermore, with CTMP as a direct binding partner of Akt1, and LETM1 as a binding partner of CTMP, LETM1-2A-CTMP downregulated the Akt1 pathway at both Ser473 and Thr308 sites of phosphorylation. Proliferation and angiogenesis, which are important in cancer prognosis, were reduced in tumor sites after introduction of LETM1-2A-CTMP. Taken together, the results indicate that introduction of the mitochondria-targeting genes, LETM1 and CTMP, and self-processing capacity of 2A-peptide sequence exerts an antitumor effect in liver of H-ras12V mice, suggesting its potential as a tool for gene therapy.

Genetic association between human chitinases and lung function in COPD.

Two primary chitinases have been identified in humans--acid mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Mammalian chitinases have been observed to affect the host's immune response. The aim of this study was to test for association between genetic variation in the chitinases and phenotypes related to chronic obstructive pulmonary disease (COPD). Polymorphisms in the chitinase genes were selected based on previous associations with respiratory diseases. Polymorphisms that were associated with lung function level or rate of decline in the Lung Health Study (LHS) cohort were analyzed for association with COPD affection status in four other COPD case-control populations. Chitinase activity and protein levels were also related to genotypes. In the caucasian LHS population, the baseline forced expiratory volume in one second (FEV(1)) was significantly different between the AA and GG genotypic groups of the AMCase rs3818822 polymorphism. Subjects with the GG genotype had higher AMCase protein and chitinase activity compared with AA homozygotes. For CHIT1 rs2494303, a significant association was observed between rate of decline in FEV(1) and the different genotypes. In the African American LHS population, CHIT1 rs2494303 and AMCase G339T genotypes were associated with rate of decline in FEV(1). Although a significant effect of chitinase gene alleles was found on lung function level and decline in the LHS, we were unable to replicate the associations with COPD affection status in the other COPD study groups.

The necessity of voiding cystourethrography in children with prenatally diagnosed hydronephrosis.

The postnatal persistence of fetal hydronephrosis requires further evaluation to establish whether pathological abnormalities are present. This study determined the necessity for voiding cystourethrography (VCUG) to identify vesicoureteral reflux (VUR) in children (n = 195) with prenatally diagnosed hydronephrosis. Among the study population, the prevalence of VUR was 17.4% (24 males, 10 females). There was a poor correlation between the severity of hydronephrosis, ureteral dilatation, presence of bilateral hydronephrosis and presence of VUR. Except for the frequency of urinary tract infections and the presence of renal damage on (99m)Tc-dimercaptosuccinic acid scans, VCUG was the only reliable method for confirming VUR in this study. The diagnosis of VUR is important for the early detection of renal damage. Further information is needed to develop the optimal approach to the evaluation of prenatal hydronephrosis, with reliable parameters that avoid invasive procedures such as VCUG.

Suppression of tumor growth in xenograft model mice by programmed cell death 4 gene delivery using folate-PEG-baculovirus.

Cancer gene therapy using tumor suppressor genes is considered to be an attractive approach for arresting cell growth and inducing apoptosis. Programmed cell death 4 (Pdcd4) is a tumor suppressor gene, which prevents tumorigenesis and tumor progression. To address the issue of whether expression of PDCD4 protein induces apoptosis in cancerous cells, the Pdcd4 gene was delivered using folate-PEG-baculovirus. Folate-PEG-baculovirus containing Pdcd4 gene (F-P-Bac-Pdcd4) was constructed by attachment of F-PEG to the baculovirus surface using chemical modification. The F-P-Bac-Pdcd4 showed enhanced transduction efficiency, efficiently expressed PDCD4 protein, and induced apoptosis in human epidermal carcinoma (KB) cells as compared with an unmodified baculovirus. In a tumor xenograft study, injection of F-P-Bac-Pdcd4 into tumors established from the KB cell line by subcutaneous implantation significantly suppressed tumor growth and induced apoptosis. Thus, this study shows a new baculovirus-mediated tumor suppressor gene delivery system for cancer therapy.

Urocanic acid-modified chitosan-mediated PTEN delivery via aerosol suppressed lung tumorigenesis in K-ras(LA1) mice.

The low efficiency of conventional therapies in achieving long-term survival of lung cancer patients calls for development of novel options. Revisiting of aerosol gene delivery may provide an alternative for safe and effective treatment for lung cancer. In this study, imidazole ring-containing urocanic acid-modified chitosan (UAC) designed in the previous study was used as a gene carrier. The potential effects of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) on Akt-related signals and cell cycle regulation were evaluated. Aerosols of UAC-PTEN were delivered into K-ras(LA1) lung cancer model mice through the nose-only inhalation system twice a week for total 4 weeks. Delivered PTEN suppressed lung tumor development significantly through nuclear complex formation between PTEN and p53, suppressing Akt-related signals as well as cell cycle regulation. Together, our results suggest that aerosol delivery of UAC-PTEN may be compatible with noninvasive in vivo gene therapy.

Lentivirus-mediated carboxyl-terminal modulator protein gene transfection via aerosol in lungs of K-ras null mice.

The low efficiency of conventional therapies in achieving long-term survival of lung cancer patients calls for development of novel options. Aerosol gene delivery may provide the alternative for safe and effective treatment for lung cancer. Therefore, current study was performed to elucidate the potential effects of C-terminal modulator protein (CTMP) via aerosol on lung tumorigenesis. Lentiviral vector-CTMP was delivered into K-ras null lung cancer mice through the nose-only inhalation system for 30 min. After 48 h, the potential effects of CTMP on Akt1-related signals and cell cycle regulation in the lungs were evaluated by western blot, immunohistochemistry and zymography. Lentivirus-based CTMP delivery inhibited the Akt1 activity through selective suppression of Akt1 phosphorylation at Ser473. Aerosol delivery of CTMP inhibited proteins important for Akt1 signals, cell cycle and tumor metastasis in lungs of K-ras null mice. Together, our results suggest that lentivirus-mediated aerosol delivery of CTMP may be compatible with noninvasive in vivo gene therapy. Our results emphasize the importance of noninvasive-targeted delivery of CTMP for lung cancer therapy in the future. While the studies are conducted in mice, it is envisioned that noninvasive targeting the specific genes responsible for cancer progression is an attractive strategy for effective anticancer therapeutics.

Aerosol-delivered programmed cell death 4 enhanced apoptosis, controlled cell cycle and suppressed AP-1 activity in the lungs of AP-1 luciferase reporter mice.

The long-term survival of lung cancer patients treated with conventional therapies remains poor and therefore the need for novel approaches remains high. This has led to the re-emergence of aerosol delivery as a therapeutic intervention. In this study, glucosylated polyethylenimine (GPEI) was used as carrier to investigate programmed cell death 4 (PDCD4) and PDCD4 mutant (D418A), an eIF4A-binding mutant, on PDCD4-related signaling and activator protein-1 (AP-1) activity in the lungs of AP-1 luciferase reporter mice. After confirming the efficiency of GPEI as a carrier in lungs, the effects of aerosol-delivered PDCD4 were investigated in AP-1 luciferase reporter mice. Aerosol delivery of GPEI/PDCD4 through a nose-only inhalation facilitated the apoptosis of lungs whereas aerosol PDCD4 mutant did not. Also, such aerosol delivery regulated proteins relevant to cell-cycle control and suppressed AP-1 activity. Results obtained by western blot analysis, immunohistochemistry, luciferase assay and deoxynucleotidyl-transferase-mediated nick end labeling study suggest that combined actions such as facilitating apoptosis, controlling cell cycle and suppression of AP-1 activity by PDCD4 may provide useful tool for designing lung tumor prevention and treatment by which PDCD4 functions as a transformation suppressor in the future.

Aerosol delivery of Akt controls protein translation in the lungs of dual luciferase reporter mice.

Lung cancer has emerged as a leading cause of cancer death in the world; however, most of the current conventional therapies are not sufficiently effective in altering the progression of disease. Therefore, development of novel treatment approaches is needed. Although several genes and methods have been used for cancer gene therapy, a number of problems such as specificity, efficacy and toxicity reduce their application. This has led to re-emergence of aerosol gene delivery as a noninvasive method for lung cancer treatment. In this study, nano-sized glucosylated polyethyleneimine (GPEI) was used as a gene delivery carrier to investigate the effects of Akt wild type (WT) and kinase deficient (KD) on Akt-related signaling pathways and protein translation in the lungs of CMV- LucR-cMyc-IRES-LucF dual reporter mice. These mice are a powerful tool for the discrimination between cap-dependent/-independent protein translation. Aerosols containing self-assembled nano-sized GPEI/Akt WT or GPEI/Akt KD were delivered into the lungs of reporter mice through nose-only-inhalation-chamber with the aid of nebulizer. Aerosol delivery of Akt WT caused the increase of protein expression levels of Akt-related signals, whereas aerosol delivery of Akt KD did not. Furthermore, dual luciferase activity assay showed that aerosol delivery of Akt WT enhanced cap-dependent protein translation, whereas a reduction in cap-dependent protein translation by Akt KD was observed. Our results clearly showed that targeting Akt may be a good strategy for prevention as well as treatment of lung cancer. These studies suggest that our aerosol delivery is compatible for in vivo gene delivery which could be used as a noninvasive gene therapy in the future.

Expression of cyclooxygenase-2 in breast carcinogenesis and its relation to HER-2/neu and p53 protein expression in invasive ductal carcinoma.

The purpose of this study was to evaluate cyclooxygenase-2 (COX-2) expression in the successive steps of breast carcinogenesis and to determine its correlation with HER-2/neu and p53 expression in invasive ductal carcinomas of the breast. Immunohistochemical staining with anti-COX-2 antibody was performed in normal breast tissue, usual hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma. Expression of COX-2 in invasive ductal carcinoma was correlated with immunohistochemical expression of HER-2/neu and p53 protein. COX-2 expression was found to be progressively elevated along the continuum from normal breast tissue to invasive ductal carcinoma (P<0.001). COX-2 expression significantly correlated with p53 and HER-2/neu protein expression (P<0.05 and P<0.001). On multivariate analysis, only TNM stage and elevated COX-2 expression correlated with survival. Our results suggest that COX-2 may be involved in the carcinogenesis of the breast and may be an independent prognostic indicator in patients with invasive ductal carcinoma. HER-2/neu and p53 are likely to be involved in the regulation of COX-2 expression in invasive ductal carcinomas of the breast.

Antioxidative effects of 7-hydroxy-3-methoxy-cadalene extracted from Zelkova serrata on 4-(methylinitros amino)-1-(3-pyridyl)-1-butanone-induced oxidative stress in A/J mice.

Effects of 7-hydroxy-3-methoxy-cadalene (cadalene) extracted from Zelkova serrata on 4-(methylinitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced oxidative stress were examined using A/J mice. NNK (65 microg/ml water) was orally administered to 20 mice for 7 weeks, followed by free feeding of a commercial diet, not containing cadalene, for 2 weeks. The control group was maintained without NNK and cadalene administration, and treatment groups with NNK and cadalene (6.25, 25, 100 mg/kg feed) feeding for 25 weeks. The glutathione concentration of cadalene-treated (65 microg/ml water) group was significantly higher than that of the group treated only with NNK (p < 0.05). The results of our study strongly indicate that cadalene exerts antioxidative effect on NNK-induced lung tumorigenesis in A/J mice.

Characterization of aberrant FHIT transcripts in gastric adenocarcinomas.

Aberrant transcripts of FHIT (fragile histidine triad) have been reported in several types of primary tumors and cell lines, including gastric carcinoma. The role of these aberrant transcripts in tumorigenesis is not clear yet. Forty-eight aberrant-sized FHIT transcripts with various lengths and number in 35 cases of gastric adenocarcinomas were further characterized. Aberrant transcripts, with deletions and/or insertions, were frequently observed in 20 cases of tumors. Sequence analysis demonstrated that different types of aberrant transcripts used normal splice sites but skipped exons, contained the inserts with the part of intron 5 sequences, or used the FHIT cDNA sequence 179-180 as a cryptic splice acceptor site. Most of aberrant transcripts lacked exon 5 and were presumably non-functional as the translation initiation codon is located in exon 5. Additionally, other transcripts, indicative of additional splice processing, either deletions or insertions, were expressed in several tumors. Taken together, our data indicate that the FHIT gene expression is frequently altered in gastric adenocarcinomas by aberrant splicing, and suggest that different types of aberrant transcripts may result during the multi-step splice processing.

Cell cycle was disturbed in the MNNG-induced initiation stage during in vitro two-stage transformation of Balb/3T3 cells.

In vitro two-stage transformation, an important method for the screening of carcinogens, is a valuable approach for the mechanistic study of multi-stage carcinogenesis. However, very little is known about the molecular and cellular mechanisms, particularly in terms of cell cycle control during in vitro two-stage transformation. We improved the in vitro two-stage transformation method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as an initiator and cadmium as a promoter, and reconfirmed the promotional effect of cadmium (Fang et al., 2001a). To determine the alterations of cell cycle control in the MNNG-induced initiation stage during transformation, we examined the effects of MNNG on Balb/3T3 A31 cell growth, and determined the alterations of the protein and/or mRNA levels of cyclins B1, D1, E, and G, PCNA, GADD45, p27, and wild-type p53. After 4 hour treatment of MNNG, populations of G2/M phase distribution and apoptotic fraction and the cyclin G mRNA level increased, while the cyclin B1 mRNA level decreased in a concentration-dependent manner. Wild-type p53, p27, and GADD45 protein levels also increased as a function of MNNG concentrations. However, cyclin D1, cyclin E, and PCNA expressions remained unchanged. During the initiation stage, PCNA protein expression decreased on the first day after MNNG-treatment, then increased gradually during the following 6 days, and further increased on the first day after cadmium treatment. Although wild-type p53 and p27 protein expressions also showed temporary retardation on the first day after MNNG-treatment, the expressions increased gradually during the following 6 days, but decreased rapidly by the cadmium treatment. These results indicated that during the initiation stage, MNNG induced G2/M arrest and apoptosis with increased expressions of wild-type p53, p27, and GADD45 proteins; and down-regulated mRNA level of cyclin B1 and up-regulated mRNA level of cyclin G. In addition, although a few of the G2/M-arrested cells proliferated gradually, most cells continued to be suppressed and inactivated by the over-expressions of wild-type p53 and p27 until the cadmium treatment.

Altered expression of the fragile histidine triad gene in primary gastric adenocarcinomas.

Genomic alterations and abnormal expression of the fragile histidine triad (FHIT) gene in gastric carcinomas were examined to determine whether the FHIT gene is actually a frequent target for alteration during gastric carcinogenesis. To correlate DNA and RNA lesions of the FHIT gene with the effect on FHIT protein expression, we have investigated the FHIT gene for loss of heterozygosity (LOH), aberrant transcripts, point mutations, and protein expression in 35 gastric adenocarcinomas. Allelic loss at D3S1300 was detected in 7 of 33 (21%) informative cases. Aberrant transcripts, with deletions and/or insertions, were observed in 20 of 35 (57.1%) cases and resulted from alternative splicing through exon skipping and/or insertion of the FHIT intron 5 sequence or activation of the cryptic splice site. Point mutations were not found in the FHIT coding region but detected in noncoding exon 2, 3, 4, or 5 of eight aberrant transcripts. Significant reduction of FHIT protein expression was observed in 22 of 35 (62.9%) cases. Aberrant FHIT transcription was shown to be associated with loss of FHIT protein expression. However, aberrant FHIT transcripts themselves were not associated with any clinicopathological parameters, such as age, sex, tumor site, or clinical stage. Moreover, there was no association between the presence of LOH at D3S1300 and the expression of aberrant FHIT transcripts. Nevertheless, high frequency of aberrant FHIT transcripts, significant rate of LOH at D3S1300, and altered expression of the FHIT protein indicate that alterations of the FHIT gene can play an important role in gastric carcinogenesis.

Improvement of in vitro two-stage transformation assay and determination of the promotional effect of cadmium.

The classical in vitro two-stage transformation method was modified for high transformation frequency, and the promotional effect of cadmium was evaluated. In this study, we reconfirmed the usefulness of the replating method and the optimal duration time between the initiator and promoter treatments for the optimal transformation of the Balb/3T3 cells. The results also showed that subsequent exposure to CdCl(2) for 2 weeks after initial exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) markedly enhanced the transformation frequency. At the concentration of 360 ng/ml, the transformation frequency was increased by 35-fold that of the cultures treated only with MNNG, and was higher than that of the positive control group treated with 100 ng/ml 12-O-tetradecanoyl phorbol-13-acetate (TPA) after MNNG treatment. This transformation frequency was higher than that reported previously. Therefore, this in vitro two-stage transformation method can be used efficiently for the screening of genotoxic and non-genotoxic carcinogens and the study of multistage carcinogenesis. These results also indicate that cadmium has a strong potency as a promoter, and the promotional effect of cadmium is higher than that of TPA.

Cadmium-induced alterations of connexin expression in the promotion stage of in vitro two-stage transformation.

During the multistage carcinogenesis, functions of several key genes involved in the cell cycle control and cell-cell communication can be damaged. Gap junction intercellular communication (GJIC) is known to transfer small, water-soluble molecules through intercellular channels composed of proteins called connexins (Cxs). Therefore, aberrant expression of Cx may be one of the critical factors for the clonal expansion of initiated cells during the two-stage transformation. We already improved the classical in vitro two-stage transformation method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as an initiator and cadmium as a promoter on Balb/3T3 A31 cells, and reconfirmed the promotional effect of cadmium with this method (Fang, M.Z., Cho, M.H., Lee, H.W., 2001. Improvement of in vitro two-stage transformation assay and detection of the promotional effect of cadmium, Toxicol. In Vitro (in press). In this study, precise roles of Cd on Cx expression in normal Balb/3T3 A31 and during the promotion stage of the in vitro two-stage transformation were elucidated. For this purpose, the Cx43, Cx32 and Cx26 protein levels, Cx43 and Cx26 mRNA levels and the cellular distribution location of Cx43 protein were determined. Normal Balb/3T3 cells expressed Cx43 and Cx32, but not Cx26. After a short-term treatment of cadmium on normal cells, phosphorylation of Cx43 protein increased and Cx32 protein level decreased. However, during the promotion stage of the in vitro two-stage transformation, transformed cells treated with cadmium for long periods expressed Cx43 and Cx32 highly, similar to the level of normal Balb/3T3 cells, compared to the nontransformed cells. Moreover, Cx43 of the transformed cells was distributed mostly in the perinuclear region rather than the intercellular membrane. These data suggest that cadmium may inhibit the GJIC by increasing the phosphorylation of Cx43 and decreasing the expression of Cx32 in the normal Balb/3T3 A31 cells. Our results also suggest that these changes are not associated with the cell transformation; transformed cells may reexpress Cx43 and Cx32 similar to the normal cells, though Cx43 protein is distributed aberrantly during the transformation process. Further studies are needed to clarify the relationship between transformation and posttranslational modification of the Cx proteins.

Apoptosis, bcl2 expression, and cell cycle analyses in nickel(II)-treated normal rat kidney cells.

Nickel compounds are carcinogenic to human and are potent inducers of kidney and lung tumors in experimental animals. In this study, the effects of nickel(II) acetate on apoptosis, cell cycle and bcl2 expression in normal rat kidney (NRK- 52E) cells were investigated. Nickel(II) induced apoptosis in NRK-52E cells as demonstrated by DNA laddering. Apparent DNA laddering was observed in cells treated with 480 microM for 48 hr. In the flow cytometric analysis using propidium iodide fluorescence, an increase of cell proportion in G2/M phase was shown in cells exposed to at least 320 microM of nickel(II) acetate, from 7.7% for 0 microM of nickel(II) to 16.5% for 480 microM of nickel(II) acetate. Induction of apoptotic cell death by nickel(II) was accompanied by reduction of bcl2 protein expression, while the level of p53 protein was not changed. Taken together, our data indicate that nickel(II)-induced apoptosis in NRK-52E cells is accompanied by G2/M cell cycle arrest, regardless of p53 function, and that bcl2-mediated signaling pathway may be involved in positive regulation of nickel(II)-induced apoptotic cell death in NRK-52E cells.

Role of corticosterone in ethyl carbamate-induced immunosuppression in female BALB/c mice.

We have recently demonstrated that the antibody response to the T-cell-dependent antigen, sheep red blood cells (SRBCs), was suppressed by ethyl carbamate in female BALB/c mice. At the same doses, ethyl carbamate decreased in the numbers of splenic macrophages, B cells, total T cells, CD4(+) T cells and CD8(+) T cells. In addition, the serum level of corticosterone was increased dose-dependently. To investigate the possible role of corticosterone in ethyl carbamate-induced immunosuppression, the antibody response to SRBCs and the subpopulation changes of splenocytes and thymocytes were determined in naive, sham-operated and adrenalectomized (ADX) female BALB/c mice. When the mice were treated intraperitoneally with 400 mg/kg ethyl carbamate, the antibody response was significantly suppressed by ethyl carbamate in naive and sham-operated mice in accompanying the decrease in spleen and thymus weights and/or the increase in the level of serum corticosterone. Meanwhile, the antibody response was not suppressed by ethyl carbamate in the ADX mice. The splenic numbers of total cells, macrophages, B and T cells, and CD4(+) cells were decreased by ethyl carbamate in naive and sham-operated mice. Meanwhile, each cell number was comparable with control in the ADX mice. The flow cytometric analyses on thymocytes did not show obvious differences as seen in the spleen. Finally, when the ADX mice were treated intraperitoneally with 25 mg/kg corticosterone, the antibody response was significantly suppressed. Taken together, our present results suggested that corticosterone might be, at least partially, responsible for ethyl carbamate-induced immunosuppression in female BALB/c mice.