Epigenetic silencing of GDF1 disrupts SMAD signaling to reinforce gastric cancer development.

Accumulating evidence reveals the effectiveness of epigenetic therapy in gastric cancer. However, the molecular mechanisms and targets underlying such therapeutic responses remain elusive. Herein, we report an aberrant yet therapeutically rectifiable epigenetic signaling in gastric carcinogenesis. Administration of DNA-demethylating drug 5-aza-2'-deoxycytidine (5-aza-dC) reduced gastric cancer incidence by ~74% (P < 0.05) in N-nitroso-N-methylurea-treated mice. Through genome-wide methylation scanning, novel promoter hypermethylation-silenced and drug-targeted genes were identified in the resected murine stomach tumors and tissues. We uncovered that growth/differentiation factor 1 (Gdf1), a member of the transforming growth factor-ß superfamily, was silenced by promoter hypermethylation in control tumor-bearing mice, but became reactivated in 5-aza-dC-treated mice (P < 0.05). In parallel, the downregulated SMAD2/3 phosphorylation in gastric cancer was revived by 5-aza-dC in vivo. Such hypermethylation-dependent silencing and 5-aza-dC-mediated reactivation of GDF1-SMAD2/3 activity was conserved in human gastric cancer cells (P < 0.05). Subsequent functional characterization further revealed the antiproliferative activity of GDF1, which was exerted through activation of SMAD2/3/4-mediated signaling, transcriptional controls on p15, p21 and c-Myc cell-cycle regulators and phosphorylation of retinoblastoma protein. Clinically, hypermethylation and loss of GDF1 was significantly associated with reduced phosphorylated-SMAD2/3 and poor survival in stomach cancer patients (P < 0.05). Taken together, we demonstrated a causal relationship between DNA methylation and a tumor-suppressive pathway in gastric cancer. Epigenetic silencing of GDF1 abrogates the growth-inhibitory SMAD signaling and renders proliferation advantage to gastric epithelial cells during carcinogenesis. This study lends support to epigenetic therapy for gastric cancer chemoprevention and identifies a potential biomarker for prognosis.

Cholinergic-receptor-independent dysfunction of mitochondrial respiratory chain enzymes, reduced mitochondrial transmembrane potential and ATP depletion underlie necrotic cell death induced by the organophosphate poison mevinphos.

Our current understanding of the nature of cell death that is associated with fatal organophosphate poisoning and the underlying cellular mechanisms is surprisingly limited. Taking advantage of the absence in an in vitro system of acetylcholinesterase, the pharmacological target of organophosphate compounds, the present study evaluated the hypothesis that the repertoire of cholinergic receptor-independent cellular events that underlie fatal organophosphate poisoning entails induction of mitochondrial dysfunction, followed by bioenergetic failure that leads to necrotic cell death because of ATP depletion. Pheochromocytoma PC12 cells incubated with the organophosphate pesticide mevinphos (0.4 or 4mumol) for 1 or 3h underwent a dose-related and time-dependent loss of cell viability that was not reversed by muscarinic (atropine) or nicotinic (mecamylamine) blockade. This was accompanied by depressed NADH cytochrome c reductase, succinate cytochrome c reductase or cytochrome c oxidase activity in the mitochondrial respiratory chain, reduced mitochondrial transmembrane potential, decreased ATP concentration, elevated ADP/ATP ratio, increased lactate dehydrogenase release and necrotic cell death. We conclude that Mev induces cholinergic receptor-independent necrotic cell death by depressing the activity of Complexes I to IV in the mitochondrial respiratory chain, eliciting reduction in mitochondrial transmembrane potential, depleting intracellular ATP contents and damaging cell membrane integrity.

Short-arm (1.9 m) +2.2 Gz acceleration: isotonic exercise load-O2 uptake relationship.

BACKGROUND: The deconditioning syndrome from prolonged bed rest (BR) or spaceflight includes decreases in maximal oxygen uptake (VO2max), muscular strength and endurance, and orthostatic tolerance. In addition to exercise training as a countermeasure, +Gz (head-to-foot) acceleration training on 1.8-2.0 m centrifuges can ameliorate the orthostatic and acceleration intolerances induced by BR and immersion deconditioning. PURPOSE: Study A was designed to determine the magnitude and linearity of the heart rate (HR) response to human-powered centrifuge (HPC) acceleration with supine exercise vs. passive (no exercise) acceleration. Study B was designed to test the hypothesis that moderate +Gz acceleration during exercise will not affect the respective normal linear relationships between exercise load and VO2max, HR, and pulmonary ventilation (VEBTPS). Study C: To determine if these physiological responses from the HPC runs (exercise + on-platform acceleration) will be similar to those from the exercise + off-platform acceleration responses. METHODS: In Study A, four men and two women (31-62 yr) were tested supine during exercise + acceleration and only passive acceleration at 100% [maximal acceleration (rpm) = Amax] and at 25%, 50%, and 75% of Amax. In Studies B and C, seven men (33+/-SD 7 yr) exercised supine on the HPC that has two opposing on-platform exercise stations. A VO2max test and submaximal exercise runs occurred under three conditions: (EX) exercise (on-platform cycle at 42%, 61%, 89% and 100% VO2max) with no acceleration; (HPC) exercise + acceleration via the chain drive at 25%,50%, and 100% Gzmax (35%, 72% and 100% VO2max); and (EXA) exercise (on-platform cycle at 42%, 61%, 89%, and 100% VO2max) with acceleration performed via the off-platform cycle operator at +2.2+/-0.2 Gz [50% of max (rpm) G]. RESULTS: Study A: Mean (+/-SE) Amax was 43.7+/-1.3 rpm (mean = +3.9+/-0.2, range = 3.3 to 4.9 Gz). Amax run time for exercise +acceleration was 50-70 s, and 40-70 s for passive acceleration. Regression of X HR on Gz levels indicated explained variances (r2) of 0.88 (exercise) and 0.96 (passive). The mean exercise HR of 107+/-4 (25%), to 189+/-13 (100%) bpm were 43-50 bpm higher (p < 0.05) than comparable passive HR of 64+/-2 to 142+/-22 bpm, respectively. Study B: There were no significant differences in VO2, HR or VEBTPS at the submaximal or maximal levels between the EX and EXA runs. Mean (+/-SE) VO2max for EX was 2.86+/-0.12 L x min(-1)(35+/-2 ml x min(-1) x kg(-1)) and for EXA was 3.09+/-0.14 L x min(-1) (37+/-2 ml-min(-1) x kg(-1)). Study C: There were no significant differences in the essentially linear relationships between the HPC and EXA data for VO2 (p = 0.45), HR (p < 0.08), VEBTPS (p = 0.28), or the RE (p = 0.15) when the exercise load was % VO2max. CONCLUSION: Addition of + 2.2 Gz acceleration does not significantly influence levels of oxygen uptake, heart rate, or pulmonary ventilation during submaximal or maximal cycle ergometer leg exercise on a short-arm centrifuge.

Recovery of Salmonella by using selenite brilliant green sulfa enrichment broth.

The efficacy and sensitivity of selenite brilliant green sulfa enrichment (SBG) broth for the isolation of Salmonella from fecal specimens were evaluated by using both clinical and artificially infected (artificial) fecal specimens. An examination of 1,588 clinical fecal specimens found Salmonella in 296 specimens, including 89 cases detected by the direct-plating xylose-lysine-desoxycholate method and an additional 207 cases detected after enrichment with SBG broth. Therefore, the recovery of Salmonella with SBG broth is increased 3.3-fold over that by the direct-plating method alone. Furthermore, the isolation rate of Salmonella is higher when using SBG broth than when using gram-negative (GN) broth or GN broth supplemented with sodium selenite. To determine the sensitivity for the recovery of Salmonella, artificial specimens containing various amounts of Salmonella were prepared and analyzed. The results indicated that the sensitivity is also higher with SBG broth than with GN broth. Moreover, the optimal incubation period for SBG broth can be extended to 24 h. In conclusion, the SBG enrichment method provides a higher recovery rate of Salmonella from fecal specimens.

Constitutive overexpression of cyclin D1 in human breast epithelial cells does not prevent G1 arrest induced by deprivation of epidermal growth factor.

Non-transformed human breast epithelial cell line MCF10A is dependent on exogenous epidermal growth factor (EGF) for continued growth. Complete G1 arrest was rapidly induced following EGF deprivation. The cell cycle arrest was accompanied by increased levels of p27KIP1, a cyclin-dependent kinase inhibitor, and reduced level of cyclin D1. This was associated with strong inhibition of cyclin-dependent kinase 2 and cyclin D1-associated kinase activities. Introduction of exogenous cyclin D1 into MCF10A (MCF10AD1) cells resulted in an accelerated cell growth rate but did not confer colony-forming capacity. Cell cycle arrest was still achieved in MCF10AD1 cells following EGF deprivation. In the great majority of MCF10AD1 clones, accumulation in G1 phase was accompanied by reduced cyclin D1 and increased p27KIP1 protein levels. In two clones where cyclin D remained unchanged during G1 arrest, it was found that more cyclin D1 protein was bound to p27KIP1. The data demonstrate that ectopic expression of cyclin D1 alone could not transform MCF10A cells nor was it sufficient to prevent G1 arrest induced by EGF deprivation.

Reciprocal changes in p27(Kip1) and p21(Cip1) in growth inhibition mediated by blockade or overstimulation of epidermal growth factor receptors.

Many human epithelial tumors express high levels of epidermal growth factor (EGF) receptors. A human-mouse chimeric version of anti-EGF receptor monoclonal antibody (mAb) C225, which blocks receptor activation and produces inhibition of cell proliferation, is currently being investigated in clinical trials. When cells bear high numbers of EGF receptors, either complete blockade of receptors with mAb 225 or full activation of receptors with EGF results in inhibition of proliferation. In the present study, we have explored the molecular mechanisms explaining how a receptor inhibitor, mAb 225, and a receptor activator, EGF, can both produce growth inhibition of A431 human squamous epithelial carcinoma cells. We reported previously that inhibition of A431 cells by EGF is associated with up-regulation of p21(Cip1). We now demonstrate that mAb 255-mediated inhibition is associated with up-regulation of p27(Kip1), which binds to and inactivates cyclin-dependent kinase-2 activity and produces cell cycle arrest in G1. Furthermore, inhibition by mAb 225 can be overcome by titrating the cultures with increasing concentrations of EGF, which is accompanied by a concurrent fall in the level of p27(Kip1). At properly titrated concentrations of mAb 225 and EGF, the inhibitory activities of both mAb 225 and EGF are counterbalanced and abolished. When EGF concentrations reach levels high enough to compete with mAb to produce near-saturating levels of receptor activation, p27(Kip1) falls below basal levels; however, the concomitant marked rise in the level of p21(Cip1) results in growth inhibition. Our data suggest that although p27(Kip1) and p21(Cip1) are induced and act independently, they play reciprocal roles in mediating inhibition of A431 cell growth by blockade of EGF receptors with mAb 225 and by activation of receptors with saturating concentrations of EGF.

Characterization of the promoter region of the cystic fibrosis transmembrane conductance regulator gene.

To identify the transcription regulatory elements of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, DNA fragments located in the 5'-upstream region were fused with the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and transfected into various cell lines to test for promoter activity. The results of these studies suggested that there were at least two positive and one negative cisacting elements involved in CFTR transcription initiation. One of them was a proximal, positive element delimited by the 5' deletion constructs -226 base parts upstream of the transcription start site. This minimal promoter sequence (-226 to +98) alone seemed to be sufficient to direct cell-specific CAT expression. The sequences immediately upstream of -227, on the other hand, appeared to contain a negative regulatory element; inclusion of this sequence with the proximal element (e.g. a construct containing sequences -345 to +98) rendered the CFTR promoter inactive. This negative regulatory element could also suppress the activity of a heterologous promoter. In addition, the DNA transfection study suggested the existence of another positive regulatory element outside the CFTR promoter region examined, as the inability of this region (e.g. -658 to +98) to function in a CAT assay could be overcome by the presence of a viral enhancer element.

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones.

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth in vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogen- or prolactin-dependent growth. Primary tumors still displayed a constitutional expression of alpha-, beta-, and gamma-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in alpha- and beta-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of alpha-, beta-, and gamma-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10-14 days. The accumulation of beta-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance beta-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.

Twenty year experience with radiation therapy for temporal bone chemodectomas.

Between 1965 and 1984, 20 patients with chemodectomas of the temporal bone were seen at The Methodist Hospital in Houston, Texas and at the Cancer Therapy and Research Center in San Antonio, Texas, Ten patients were treated with radiation therapy alone, seven with surgery and post-operative radiation, one with pre-operative radiation, and two with radiation therapy following surgical recurrence. Most patients had advanced tumors at presentation. Radiation doses ranged from 22.5 Gy to 50.0 Gy. The most frequent dose was 45.0 Gy, given in 225 cGy fractions, 9.0 Gy per week. The most common radiation portal arrangement was oblique fields with paired wedges. There were no local failures or significant radiation induced complications among the patients with benign chemodectomas. The follow-up period ranged from 3 to 23 years (mean 11 years). Only one patient developed systemic metastases and progression of the primary temporal bone chemodectoma. These results and a review of the literature demonstrate that radiation therapy alone is a safe and effective treatment modality for chemodectomas of the temporal bone.

The treatment of advanced juvenile nasopharyngeal angiofibroma.

Fifteen patients with juvenile nasopharyngeal angiofibroma (JNA) were treated in the Department of Radiation Oncology, Baylor College of Medicine between 1973 and 1986. All patients underwent radiographic evaluation including CT scanning, selective digital subtraction angiography, tomograms, or MRI. Patients referred for definitive irradiation exhibited extensive tumor involvement. Eleven of 15 patients had middle cranial fossa involvement; cavernous sinus extension was observed in six patients. Ten patients were treated with primary radiation therapy; five patients had surgical resection initially and were referred for radiation therapy upon local recurrence. Follow-up ranges from 1 1/2-13 years. Four of the 5 patients who received 3200 cGy in 200 cGy fractions demonstrated tumor recurrence within 2 years after irradiation. All recurrences were ultimately controlled by either further irradiation and/or resection. No tumor recurrence was encountered among the patients treated at the higher tumor doses (36-46 Gy). No severe complications have been observed. Radiation therapy utilizing carefully tailored fields is an appropriate therapeutic approach to patients with extensive disease or intracranial extension. A total dose of greater than 40 Gy may allow improved local control for advanced lesions.

Effects of extracellular matrix on the growth and casein gene expression of primary mouse mammary tumor cells in vitro.

This study describes a serum-free culture system and provides a tumor model to investigate the effects of extracellular matrices on the growth and beta-casein gene expression of mouse mammary tumor epithelial cells (MMTCs) in vitro. Primary cultures of MMTCs derived from autochthonous mammary tumors in BALB/cfC3H x DBA/8 F1 mice, FUKU cells, an established MMTC line, and COMMA-1D cells established from mouse mammary tissues were studied. A reconstituted basement membrane from the Englbreth-Holm-Swarm tumor (Matrigel) allowed a 2.7-fold increase in cell number of 5-day primary MMTC cultures in serum-free, insulin-supplemented medium. FUKU and COMMA-1D cells in serum-free medium displayed a 13.6- and 11.5-fold increase in cell number, respectively, after 5 and 6 days in culture on Matrigel. In semisolid agar cultures, Matrigel or laminin was shown to promote colony-forming efficiency of FUKU cells when either of the matrices was mixed in the top agar layer. An increase of 4.4 or 2.1 times in colony-forming efficiency was detected when 20% (v/v) Matrigel or 112 micrograms/ml of laminin were mixed in the agar layer compared with FUKU cells plated in plain agar. beta-Casein mRNA was detectable by Northern blot assays in the primary mammary tumors. MMTCs in primary cultures grown on Matrigel in serum-free, insulin-supplemented medium for 4 days were inducible for beta-casein mRNA following the treatment with prolactin and hydrocortisone (FPRL) for 24 h. No beta-casein mRNA was detectable in the absence of FPRL. MMTCs in the primary cultures could also be induced for beta-casein mRNA when they were cultivated on type I collagen gels for 4 days but not on laminin, type IV collagen, or plastic. However, the capacity to respond to FPRL was not lost in MMTCs cultured on laminin. When MMTCs were initially cultured on laminin for 4 days and then subcultured on Matrigel for another 4 days, they were inducible for beta-casein mRNA upon exposure to FPRL for 24 h. In contrast, no beta-casein mRNA upon exposure to FPRL for 24 h. In contrast, no beta-casein mRNA was found in MMTCs from the same tumors cultured on laminin for 8 days with the same treatment of hormones. These data demonstrate that cells from autochthonous mammary tumors, which are not dependent on estrogen for growth in vivo, are inducible in vitro for beta-casein mRNA by FPRL; and this hormonal response of MMCTs requires appropriate extracellular matrix.

Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.

Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7. Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF. The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding. A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.

Effective radiotherapy in palliating mammalgia associated with gynecomastia after DES therapy.

Between 1977 and 1986, 11 patients with painful gynecomastia after DES therapy were referred for palliative radiotherapy. The treatment regimens varied from 20 Gy in 5 fractions to 40 Gy in 20 fractions. All 11 patients had satisfactory pain relief on follow-up. All 7 patients who had more than 6 months follow-up had complete relief of mammalgia. The average interval between completion of radiotherapy to complete relief of mammalgia was 3.6 months. This study revealed that radiotherapy is highly effective in palliating mammalgia associated with gynecomastia after DES therapy in prostate cancer patients.

Analysis of human peripheral blood T lymphocytes proliferating in response to sensitizing antigens: effect of interleukin-2 (IL-2)-responsive bystander cells.

The contribution of interleukin-2 (IL-2)-responsive bystander cells to the proliferative responses of human peripheral blood T lymphocytes to antigens used for sensitization such as Purified Protein Derivative (PPD), Tetanus toxoid (T T) and Influenza virus was investigated. Marked proliferation of the unfractionated peripheral blood mononuclear cells (PBMC) was observed following stimulation with these antigens to which the individuals were known to have been sensitized previously. Depletion of large granular lymphocytes (LGL) from PBMC resulted in substantial reduction in the response of the lymphoid cells in proliferating to the antigens. Proliferation of the T4+T8- (helper)-enriched population, or T4+T8- subset depleted of any IL-2 receptor (IL-2R)-bearing lymphoid cells to these antigens was comparable to that of LGL-depleted PBMC cultures. Cell titration experiments of the blast cells generated from these cultures revealed that PBMC-derived population contained fewer antigen-reactive lymphocytes. These results, therefore, suggested that IL-2-responsive LGL through expansion affected the concentration of antigen-proliferating T cells in the antigen-stimulated PBMC cultures.

Controversy in the management of optic pathway gliomas. 29 patients treated with radiation therapy at Baylor College of Medicine from 1967 through 1987.

The optic gliomas of 29 patients, including 14 with von Recklinghausen neurofibromatosis (NF-1), were subjected to X-ray therapy. The data indicate a projected 20-year survival rate of 92% for all 29 patients. Moreover, among the NF-1 patients, 86% were stabilized or improved, while among non-NF-1 patients, only 47% stabilized or were improved. Thus, these data suggest that there are differences in the biophysiological behavior of optic nerve gliomas in patients with NF-1, and, as well, that there is a salutary response to radiation treatment as measured by improvement or stabilization of vision, with and without radiologic evidence of concomitant tumor regression.

In vivo activated lymphoid cells (IVALC) affect the cloning efficiency of human T lymphocytes reactive to a soluble antigen, purified protein derivative.

Peripheral blood mononuclear cells (PBMC) of normal individuals were found to contain a proportion (4-9%) of in vivo activated lymphoid cells (IVALC). These IVALC were characterized by their expression of interleukin 2 (IL-2) receptors, and by the ability to proliferate in the presence of exogenous IL-2. There was a good correlation between the proportion of IVALC in different cell populations and the level of cell proliferation to IL-2. It was found that IVALC isolated from autologous PBMC of Bacillus Calmette-Guerin (BCG)-immunized individuals contained no significant proportion of purified protein derivative (PPD)-reactive lymphocytes. The addition of IVALC markedly enhanced proliferative responses of the autologous T4+T8-IL-2 receptor-negative cell cultures to antigen stimulation. An increased proportion of activated (IL-2 receptor-positive) lymphocytes was generated in PBMC as compared to autologous T4+T8-IL-2 receptor negative cell cultures after stimulation with PPD. Limiting dilution analysis showed that IL-2 responsive IVALC through expansion markedly affected the cloning efficiency of antigen-proliferating T cells of autologous PPD-stimulated PBMC cultures. Only 1 out of every 11-25 blast cells generated in the PBMC cultures could establish itself as a growing colony based on determinations in six BCG-positive individuals. By using a T4+T8- population depleted of IVALC to generate PPD-reactive lymphocytes, a three- to four-fold increase in the cloning efficiency of antigen-specific cells was obtained.

Phenotypic modulation of T-lymphoblastic leukaemic cells with phorbol ester and leucocyte conditioned medium.

Human T-lymphoblastic leukaemic cell line Karpas-45 (K-45) can be induced by phorbol 12-myristate 13-acetate (PMA) to become phenotypically more mature. Marker studies show that the percentages of E-rosette-positive (E+) and UCHT1+ cells are increased after exposure to PMA. Fluorescence of UCHT4+ and 2D1+ cells is increased although their percentages remain almost unchanged. OKT4+ and peanut agglutinin (PNA)-positive cells are greatly reduced. Terminal deoxynucleotidyl transferase (TdT) becomes negative. PMA treatment of K-45 cells after deletion of UCHT4+ cells suggest that marker changes detected by UCHT2, OKT4 and sheep red blood cells (SRBC) occur mainly in the UCHT4+ cells. The proliferation of PMA-treated K-45 cells is dramatically inhibited and the cell size becomes smaller. These results indicate that K-45 cells are induced to differentiate phenotypically towards a suppressor/cytotoxic T cell. However functional maturation can not be demonstrated. Phytohemagglutinin-stimulated leucocyte conditioned medium (LCM) is able to suppress DNA synthesis of K-45 cells and reduce the PNA+ proportion. It seems that LCM can induce a limited degree of differentiation of K-45 cells.