Increased C-myc mRNA Research Articles

Lysyl oxidase inhibits BMP9-induced osteoblastic differentiation through reducing Wnt/β-catenin via HIF-1a repression in 3T3-L1 cells

BackgroundBone morphogenetic protein 9 (BMP9) is a promising growth factor in bone tissue engineering, while the detailed molecular mechanism underlying BMP9-oriented osteogenesis remains unclear. In this study, we investigated the effect of lysyl oxidase (Lox) on the BMP9 osteogenic potential via in vivo and in vitro experiments, as well as the underlying mechanism.MethodsPCR assay, western blot analysis, histochemical staining, and immunofluorescence assay were used to quantify the osteogenic markers level, as well as the possible mechanism. The mouse ectopic osteogenesis assay was used to assess the impact of Lox on BMP9-induced bone formation.ResultsOur findings suggested that Lox was obviously upregulated by BMP9 in 3T3-L1 cells. BMP9-induced Runx2, OPN, and mineralization were all enhanced by Lox inhibition or knockdown, while Lox overexpression reduced their expression. Additionally, the BMP9-induced adipogenic makers were repressed by Lox inhibition. Inhibition of Lox resulted in an increase in c-Myc mRNA and β-catenin protein levels. However, the increase in BMP9-induced osteoblastic biomarkers caused by Lox inhibition was obviously reduced when β-catenin knockdown. BMP9 upregulated HIF-1α expression, which was further enhanced by Lox inhibition or knockdown, but reversed by Lox overexpression. Lox knockdown or HIF-1α overexpression increased BMP9-induced bone formation, although the enhancement caused by Lox knockdown was largely diminished when HIF-1α was knocked down. Lox inhibition increased β-catenin levels and decreased SOST levels, which were almost reversed by HIF-1α knockdown.ConclusionLox may reduce the BMP9 osteoblastic potential by inhibiting Wnt/β-catenin signaling via repressing the expression HIF-1α partially.Graphical abstract

LINC01123, a c-Myc-activated long non-coding RNA, promotes proliferation and aerobic glycolysis of non-small cell lung cancer through miR-199a-5p/c-Myc axis

BackgroundLong non-coding RNAs (lncRNAs) have been associated with non-small cell lung cancer (NSCLC), but the underlying molecular mechanisms of their specific roles in mediating aerobic glycolysis have been poorly explored.MethodsNext-generation RNA sequencing assay was performed to identify the differentially expressed RNAs between NSCLC tissues with high 18F-fluorodeoxyglucose (FDG) uptake and their adjacent normal lung tissues. LINC01123 expression in NSCLC tissues was measured by real-time PCR and in situ hybridization (ISH) assay. The biological role of LINC01123 in cell growth and aerobic glycolysis capability was determined by performing functional experiments in vitro and in vivo. Further, the transcription of LINC01123 was explored by bioinformatics analysis, dual-luciferase reporter assay, and chromatin immunoprecipitation (ChIP) assay. RNA immunoprecipitation (RIP) and luciferase analyses were used to confirm the predicted competitive endogenous RNA (ceRNA) mechanisms between LINC01123 and c-Myc.ResultsThree hundred sixty-four differentially expressed genes were identified in RNA-seq assay, and LINC01123 was one of the most overexpressed lncRNAs. Further validation in expanded NSCLC cohorts confirmed that LINC01123 was upregulated in 92 paired NSCLC tissues and associated with poor survival. Functional assays showed that LINC01123 promoted NSCLC cell proliferation and aerobic glycolysis. Mechanistic investigations revealed that LINC01123 was a direct transcriptional target of c-Myc. Meanwhile, LINC01123 increased c-Myc mRNA expression by sponging miR-199a-5p. In addition, rescue experiments showed that LINC01123 functioned as an oncogene depending on miR-199a-5p and c-Myc.ConclusionSince LINC01123 is upregulated in NSCLC, correlates with prognosis, and controls proliferation and aerobic glycolysis by a positive feedback loop with c-Myc, it is expected to be a potential biomarker and therapeutic target for NSCLC.

Effects of testosterone treatment on markers of skeletal muscle ribosome biogenesis.

The effects of testosterone (TEST) treatment on markers of skeletal muscle ribosome biogenesis invitro and invivo were examined. C2 C12 myotubes were treated with 100nm TEST for short-term (24-h) and longer-term (96-h) treatments. Moreover, male 10-month-old Fischer 344 rats were housed for 4weeks, and the following groups were included in this study: (i) Sham-operated (Sham) rats, (ii) orchiectomised rats (ORX) and (iii) ORX+TEST-treated rats (7.0mgweek-1 ). For invitro data, TEST treatment increased c-Myc mRNA expression by 38% (P=0.004) after 96h, but did not affect total RNA, 47S pre-rRNA, Raptor mRNA, Nop56 mRNA, Bop1 mRNA, Ncl mRNA at 24h or 96h following the treatment. For invivo data, ORX decreased levator ani/bulbocavernosus (LABC) myofibril protein versus Sham (P=0.006), whereas ORX+TEST (P=0.015) rescued this atrophic effect. ORX also decreased muscle ribosome content (total RNA) compared to Sham (P=0.046), whereas ORX+TEST tended to rescue this effect (P=0.057). However, other markers of ribosome biogenesis including c-Myc mRNA, Nop56 mRNA, Bop1 mRNA, Ncl mRNA decreased with ORX independently of TEST treatments (P<0.05). Finally, lower phospho-(Ser235/236)-to-total rps6 protein and lower rpl5 protein levels existed in ORX+TEST rats versus other treatments, suggesting that chronic TEST treatment may lower translational capacity.

C-Myc phosphorylation by PKCζ represses prostate tumorigenesis

Studies showing reduced PKCζ expression or enzymatic activity in different types of human cancers support the clinical relevance of PKCζ as a tumor suppressor. However, the in vivo role of PKCζ and its mechanisms of action in prostate cancer remain unclear. Here we demonstrate that the genetic inactivation of PKCζ in mice results in invasive prostate carcinoma in vivo in the context of phosphatase and tensin homolog deficiency. Bioinformatic analysis of human prostate cancer gene-expression sets revealed increased c-Myc transcriptional activity in PKCζ-inactive cells, which correlated with increased cell growth, invasion, and metastasis. Interestingly, PKCζ knockdown or the overexpression of a kinase-inactive mutant resulted in enhanced cell proliferation and invasion in vitro through increased c-Myc mRNA and protein levels and decreased Ser-373 phosphorylation of c-Myc. Analysis of prostate cancer samples demonstrated increased expression and decreased phosphorylation of c-Myc at Ser-373 in PKCζ knockout tumors. In vivo xenograft studies revealed that c-Myc phosphorylation by PKCζ is a critical event in the control of metastasis. Collectively, these results establish PKCζ as an important tumor suppressor and regulator of c-Myc function in prostate cancer.

Genome-wide CpG island methylation analysis implicates novel genes in the pathogenesis of renal cell carcinoma

In order to identify novel candidate tumor suppressor genes (TSGs) implicated in renal cell carcinoma (RCC), we performed genome-wide methylation profiling of RCC using the HumanMethylation27 BeadChips to assess methylation at >14,000 genes. Two hundred and twenty hypermethylated probes representing 205 loci/genes were identified in genomic CpG islands. A subset of TSGs investigated in detail exhibited frequent tumor methylation, promoter methylation associated transcriptional silencing and reactivation after demethylation in RCC cell lines and down-regulation of expression in tumor tissue (e.g., SLC34A2 specifically methylated in 63% of RCC, OVOL1 in 40%, DLEC1 in 20%, TMPRSS2 in 26%, SST in 31% and BMP4 in 35%). As OVOL1, a putative regulator of c-Myc transcription, and SST (somatostatin) had not previously been linked to cancer and RCC, respectively, we (1) investigated their potential relevance to tumor growth by RNAi knockdown and found significantly increased anchorage-independent growth and (2) demonstrated that OVOL1 knockdown increased c-Myc mRNA levels.

Downregulation of c-Myc in pterygium and cultured pterygial cells

The aim of this study was to determine c-Myc expression in pterygial tissue and in cultured fibroblasts and epithelial cells of pterygia, using normal human conjunctival samples as a control. Expression of c-Myc in pterygium and normal human conjunctiva tissue was examined by immunohistochemical assay and Western blot. Fibroblasts and epithelial cells from primary pterygium and normal human conjunctiva were cultured in medium with or without transforming growth factor beta1 for up to 3 days. c-Myc protein expression was analysed by indirect immunofluorescence. Levels of c-Myc mRNA in fibroblasts were measured by reverse transcription polymerase chain reaction. c-Myc protein expression was reduced in human pterygial tissue compared with normal human conjunctiva in immunohistochemical staining. The decrease was further verified by Western blot analysis. In primary cultured cells c-Myc expression as stained by immunofluorescence was reduced in both pterygial epithelial and fibroblast cells when compared with cultured normal human conjunctiva cells. In addition, compared with cells from normal conjunctiva, reduced levels of c-Myc mRNA were observed in both cultured pterygial epithelial and fibroblast cells. Increased c-Myc expression in pterygial cells, but not in normal conjunctival cells, was found when the cultured cells were treated with transforming growth factor beta1. This increase of c-Myc mRNA appeared to be time and transforming growth factor beta1 dose dependent. c-Myc expression is reduced in pterygium compared with normal conjunctiva. Transforming growth factor beta1 can elevate the downregulated c-Myc in cultured pterygial cells.

Analysis of the protein expression changes during taxol-induced apoptosis under translation inhibition conditions

Taxol is currently used in chemotherapeutic treatments of different types of cancers. In this article, we demonstrate that taxol induces apoptosis and translation down-regulation in human embryonic kidney (HEK293T) cells. Antibody arrays are a promising new tool for the analysis of protein levels changes in cells responding to different stimuli. Using this approach, we have identified changes in the expression of 38 proteins (20 down-regulated and 18 up-regulated), implicated in several cellular processes mainly in apoptosis, cell cycle and signal transduction pathways, and also cytoskeleton proteins. Among them, we have confirmed a considerable decrease in the expression of p14(ARF) and a significant increase in the levels of dystrophin and c-Myc. It is known that c-Myc mRNA has an internal ribosome entry segment (IRES) element in its 5'UTR that could regulate its expression under global protein synthesis inhibition conditions. We demonstrate that after taxol treatment, the c-Myc IRES activity is maintained meanwhile cap-dependent activity is inhibited. In addition, an increase in c-Myc mRNA was also observed after taxol treatment. We conclude that taxol-induced c-Myc expression is regulated at both transcriptional and translational levels, the last of them by a mechanism mediated by IRES.

TNF‐α induced alterations in the Wnt signaling cascade: a potential mechanism for obesity‐associated colorectal tumorigenesis

Epidemiological studies consistently implicate obesity as an important risk factor for several types of cancer, and the data is particularly compelling for colorectal cancer. However, the underlying mechanism is not defined. This study investigated whether Wnt‐signaling, a critical pathway in colorectal carcinogenesis, is upregulated by the inflammatory cytokine, TNF‐α, which has been found to be elevated in obesity. Obesity was induced in C57BL6 mice using a high‐fat diet (60% kcal). TNF‐α protein in the colon, determined by ex vivo culture of the colon followed by ELISA of the media, was significantly elevated (p&lt;0.05) in obese mice. Phospho‐GSK3β, which is an important intermediary in Wnt signaling and which has been reported to be induced by TNF‐α, was elevated by immunohistochemistry in the colonic mucosa of obese mice. Significant elevations of β‐catenin protein as well as increased c‐myc mRNA were evident in the colonic mucosa of the obese mice (p&lt;0.05), and are also consistent with Wnt activation. These results demonstrate that obesity elevates colonic levels of TNF‐α and provide evidence that it may activate Wnt‐signaling in that organ. (This material is based on work supported by the USDA under agreement no. 58‐1950‐7‐707 and by the Prevent Cancer Foundation)

C-myc Promoter Binding Protein Regulates the Cellular Response to an Altered Glucose Concentration

Alpha-enolase is a bifunctional gene encoding both a glycolytic enzyme and a DNA binding protein, c-myc binding protein (MBP-1). MBP-1 binds the c-myc promoter and downregulates c-myc transcription. Since these alpha-enolase gene products have important functions in glucose metabolism and growth regulation, this gene may play a central role in regulating the abnormal proliferative characteristics of transformed cells. To determine the role of alpha-enolase and MBP-1 in the cellular response to altered exogenous glucose concentration, MCF-7 cells were cultured in low (1 nM), physiological (5 mM), or high (25 mM) levels of glucose. Levels of alpha-enolase, MBP-1, and c-myc expression were compared to levels of cell proliferation and lactate production. At all glucose concentrations, MCF-7 cells demonstrated an initial increase in MBP-1 expression and a parallel decrease in c-myc transcript levels, which were accompanied by decreased proliferation. Cells grown in low glucose maintained the increased MBP-1 expression through 48 h, resulting in persistently lower rates of proliferation. However, physiologic or high glucose levels resulted in decreased MBP-1 expression, which was associated with increased cellular proliferation and lactate production. In these cells, c-myc mRNA returned to control levels as MBP-1 expression decreased. Cells grown in low glucose demonstrated a dramatic increase in c-myc mRNA at 48 h, which was associated with a loss in c-myc P2 promoter binding by MBP-1. This suggests that post-translational modifications of MBP-1 likely alter its DNA binding activity. These results demonstrate an important role for MBP-1 in the altered cell proliferation and energy utilization that occur in response to an altered glucose concentration.

Identification of clinically-informative biomarkers for risk stratification within the spectrum of gastro-esophageal reflux disease in the South African population

Background : Barrett's esophagus is the precursor of esophageal adenocarcinoma with a 5-year survival rate of 25-30%. Objective : To define clinically useful biomarkers for transcriptional profiling in South African patients with Barrett's esophagus in order to identify those patients with an increased cancer risk necessitating intensified surveillance intervals. Materials and method : One-hundred and two patients were recruited for the study. COX-2, c-myb and c-myc mRNA expression were measured using a quantitative PCR method in endoscopically obtained specimens of Barrett's metaplasia (BM, n=26) or dysplasia (BD, n=14) and matching squamous esophageal tissue (n=40), squamous esophageal tissue of patients with erosive esophagitis (n=20), non-erosive esophagitis (n=20) and normal controls (n=20). Two patients with Barrett's adenocarcinoma (BAC) were also studied. Results : Demographic data of the groups were comparable. No significant differences in m-RNA expression levels were observed between ethnic groups for the genes analyzed. In the BD/BAC group 69% (11/16) showed increased c-myb m-RNA expression compared with 35% (9/26) in the BM group (p = 0.03). In the BD patients 19% (3/16) had increased c-myc m-RNA expression compared to none in those with BM and BAC. One patient each with BM and BAC had increased COX-2 m-RNA levels. No significant associations were observed for COX-2 in any of the other study groups. Conclusion : In the South African study cohort c-myb appears to be a clinically useful molecular marker for Barrett's esophagus and increased cancer risk, since m-RNA levels are progressively more over expressed in the metaplasia-dysplasia-adenocarcinoma sequence. Further studies are required to clarify the potential significance c-myc and COX-2 in South African patients with Barrett's esophagus. South African Gastroenterology Review Vol. 3(1) 2005: 5-9

Genetic heterogeneity of the MYC oncogene in advanced juvenile angiofibromas

Despite their benign histological appearance, juvenile angiofibromas sometimes exhibit an aggressive growth behavior. Molecular and genetic analyses have detected β-catenin mutations and androgen receptor gene gains in this tumor. Because intensive cross-talk among β-catenin, androgen receptor, and C-MYC has been detected recently, we analyzed expression of the C-MYC protooncogene ( MYC) on the genetic, transcriptional and translational level in seven sporadic juvenile angiofibromas. Two-color in situ hybridization analyses for chromosome 8 and MYC found in all seven juvenile angiofibromas significant MYC losses. In the three advanced juvenile angiofibromas of this series (Fisch stages III and IV) additional significant MYC gains were observed demonstrating a genetic heterogeneity for the MYC protooncogene. In cases of genetic MYC heterogeneity, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, Western blot investigations, and immunohistology showed increased C-MYC mRNA and protein levels. Semiquantitative RT-PCR analyses from laser microdissected endothelial cells and fibroblasts found no differences of C-MYC mRNA levels, leaving open the question of the neoplastic cell in juvenile angiofibromas. The finding of genetic MYC heterogeneity associated with C-MYC overexpression on the mRNA and protein level in advanced juvenile angiofibromas indicates involvement of the MYC oncogene in aggressive growth behavior.

Downregulation of cytochromes P450 in growth-stimulated rat hepatocytes: role of c-Myc induction and impaired C/EBP binding to DNA

Background/Aims: Several cytochromes P450 (CYPs) are expressed in differentiated hepatocytes, but downregulated in growth-stimulated cells. We determined the signals involved in CYP downregulation by epidermal growth factor (EGF). Methods: Rat hepatocytes were cultured with or without diverse substances for 72 h and EGF for the last 48 h. Results: EGF increased c-myc mRNA and protein, and decreased CYP mRNAs and proteins; both effects were prevented by two agents blocking c-myc transcription (retinoic acid and DMSO) and two antisense c-myc oligomers. Despite unchanged CCAAT-enhancer binding protein α (C/EBPα) and increased C/EBPβ levels, nuclear proteins of EGF-treated cells did not bind to a C/EBP DNA probe in a gel mobility shift assay. This binding was restored when cells were co-treated with both EGF and c-myc antisense oligomers (preventing c-Myc induction). The N-terminal c-Myc domain added to control nuclear extracts prevented C/EBP DNA binding. A monoclonal anti-c-Myc antibody co-immunoprecipitated c-Myc, C/EBPα and C/EBPβ from nuclear extracts. In cells not treated with EGF, an antisense C/EBPα oligomer decreased CYP expression. Conclusions: EGF overexpresses c-Myc, decreases C/EBP binding to DNA and downregulates CYPs. We suggest that c-Myc may form inactive complexes with C/EBPs, thus decreasing C/EBP-mediated CYP transactivation.

Potential role of the early response gene c-myc in beta-cell adaptation to changes in glucose concentration.

Type 2 diabetes is characterized by a reduction in both insulin sensitivity and glucose-stimulated insulin secretion (GSIS). Although insulin resistance usually precedes � -cell dysfunction, the role of the latter in the development of the disease is unclear. The detrimental effect that chronic hyperglycemia exerts on � -cell function is well established and may contribute to further deterioration of GSIS during the development of type 2 diabetes. Islets from hyperglycemic 90%pancreatectomized rats display profound alterations in GSIS and � -cell gene expression. The association of these changes with � -cell hypertrophy and increased mRNA levels of the early response gene c-myc could reflect a loss of � -cell differentiation (1). The complete reversal of all these abnormalities upon correction of hyperglycemia by phlorizin treatment suggested hyperglycemia as their triggering factor. The aim of this study was to determine whether glucose directly regulates islet c-myc expression. Rat islets were cultured for 1‐7 days in RPMI medium containing 5, 10, 20, or 30 mmol/l glucose (G5‐G30) and 0.5% bovine serum albumin. Islet c-myc and TATA box binding protein (TBP) mRNA levels were measured by semiquantitative reverse transcriptase‐polymerase chain reaction. As compared with freshly isolated islets from the same preparation, the ratio of c-myc to TBP was increased approximately fivefold after overnight culture. This increase persisted after 3‐7 days in G5. In contrast, the c-myc‐to‐TBP ratio returned to control values after 7 days in G10‐G30. To avoid the confounding effect of the early increase in c-myc mRNA, probably due to stress of isolation, islets were first cultured for 7 days in G10 and then overnight in G5‐G30. When the glucose concentration was kept at 10 mmol/l, the c-myc‐to‐TBP ratio remained low and stable, whereas an approximate fivefold increase was observed after 1 day at either lower or higher glucose. In contrast, c-myc heterodimerization partner Max and its partner Mad1 were not affected by culture or glucose. The increase in islet c-myc‐to‐TBP ratio produced by high glucose was confirmed in rats maintained hyperglycemic (blood glucose >11 mmol/l) for 1‐4 days by glucose clamp (~200% of saline-infused rats). In contrast, fasting for 3 days reduced blood glucose to 3 mmol/l and modestly increased islet c-myc‐to‐TBP ratio to 132% of the ratio in fed rats. These results strongly suggest that both low and high glucose increase islet c-myc expression. Whereas the increase in low glucose could be related to the higher rate of apoptosis reported under this condition, the increase produced by high glucose could play a role in � -cell mass adaptation to hyperglycemia (replication/hypertrophy).

HCaRG, a Novel Calcium-regulated Gene Coding for a Nuclear Protein, Is Potentially Involved in the Regulation of Cell Proliferation

Since a negative calcium balance is present in spontaneously hypertensive rats, we searched for the gene(s) involved in this dysregulation. A cDNA library was constructed from the spontaneously hypertensive rat parathyroid gland, which is a key regulator of serum-ionized calcium. From seven overlapping DNA fragments, a 1100-base pair novel cDNA containing an open reading frame of 224 codons was reconstituted. This novel gene, named HCaRG (hypertension-related, calcium-regulated gene), was negatively regulated by extracellular calcium concentration, and its basal mRNA levels were higher in hypertensive animals. The deduced protein showed no transmembrane domain, 67% alpha-helix content, a mutated calcium-binding site (EF-hand motif), four putative "leucine zipper" motifs, and a nuclear receptor-binding domain. At the subcellular level, HCaRG had a nuclear localization. We cloned the human homolog of this gene. Sequence comparison revealed 80% homology between rats and humans at the nucleotide and amino acid sequences. Tissue distribution showed a preponderance in the heart, stomach, jejunum, kidney (tubular fraction), liver, and adrenal gland (mainly in the medulla). HCaRG mRNA was significantly more expressed in adult than in fetal organs, and its levels were decreased in tumors and cancerous cell lines. We observed that after 60-min ischemia followed by reperfusion, HCaRG mRNA declined rapidly in contrast with an increase in c-myc mRNA. Its levels then rose steadily to exceed base line at 48 h of reperfusion. HEK293 cells stably transfected with HCaRG exhibited much lower proliferation, as shown by cell count and [(3)H]thymidine incorporation. Taken together, our results suggest that HCaRG is a nuclear protein potentially involved in the control of cell proliferation.

Epidermal growth factor and insulin-induced deoxyribonucleic acid synthesis in primary rat hepatocytes is phosphatidylinositol 3-kinase dependent and dissociated from protooncogene induction.

The mitogenic response to insulin and epidermal growth factor (EGF) was studied in subconfluent and confluent cultures of primary rat hepatocytes. In subconfluent cultures, wortmannin, LY294002, and rapamycin reversed insulin- and EGF-induced [3H]thymidine incorporation into DNA. The mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) inhibitor PD98059 was without significant effect on either insulin- or EGF-induced [3H]thymidine incorporation. Insulin treatment did not alter levels of messenger RNAs (mRNAs) for c-fos, c-jun, and c-myc. EGF induced an increase in c-myc, but not c-fos or c-jun, mRNA levels in subconfluent hepatocyte cultures. This increase in c-myc mRNA was abolished by PD98059. In confluent cells that could not be induced to synthesize DNA, EGF treatment also promoted an increase in c-myc mRNA to levels seen in subconfluent cultures. This increase was also abrogated by PD98059. These data indicate that in primary rat hepatocyte cultures, 1) the phosphoinositol 3-kinase pathway, perhaps through p70s6k activation, regulates DNA synthesis in response to insulin and EGF; 2) the MAPKpathway is not involved in insulin- and EGF-induced DNA synthesis; and 3) p44/42 MAPKs are involved the induction of c-myc mRNA levels, although this induction is not required for DNA synthesis. These studies define two distinct signal transduction pathways that independently mediate growth-related responses in a physiologically relevant, normal cell system.

Differential apoptosis by indomethacin in gastric epithelial cells through the constitutive expression of wild-type p53 and/or up-regulation of c-myc

Nonsteroidal anti-inflammatory drug (NSAID)-induced apoptosis is considered to be an important mechanism in the antineoplastic effects and damage produced by the drugs in the gastrointestinal tract. In this study, two different gastric cancer cell lines, MKN28 (mutant-type p53) and AGS (wild-type p53), were compared as to growth inhibition, apoptosis, and cell cycle and apoptosis-related gene expression in response to indomethacin treatment. Cell growth was measured by MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Apoptosis was characterized by acridine orange staining and DNA fragmentation, and cell cycle kinetics by flow cytometry. The mRNA and protein levels of p53, p21 waf1/cip1, and c-myc were determined by Northern and Western blotting. The results showed that indomethacin initiated growth inhibition and apoptosis in both cell lines without cell cycle shifting. AGS cells were more sensitive to growth inhibitory activity and apoptosis of indomethacin than MKN28 cells. In MKN28 cells, the levels of p53, p21 waf1/cip1, and c-myc mRNA remained unchanged over the 24-hr treatment with indomethacin, but the p53 protein level was elevated after 4 hr. There was no change in the p21 waf1/cip1 and c-myc protein levels in the MKN28 cells. In AGS cells, a progressive increase in c-myc mRNA and protein levels was noted, while p53 and p21 waf1/cip1 remained unchanged. It can be concluded that wild-type p53 and/or up-regulation of c-myc is associated with indomethacin-mediated differential apoptosis in gastric epithelial cells.

Chronic Hyperglycemia Triggers Loss of Pancreatic β Cell Differentiation in an Animal Model of Diabetes

Differentiated pancreatic beta cells are unique in their ability to secrete insulin in response to a rise in plasma glucose. We have proposed that the unique constellation of genes they express may be lost in diabetes due to the deleterious effect of chronic hyperglycemia. To test this hypothesis, Sprague-Dawley rats were submitted to a 85-95% pancreatectomy or sham pancreatectomy. One week later, the animals developed mild to severe chronic hyperglycemia that was stable for the next 3 weeks, without significant alteration of plasma nonesterified fatty acid levels. Expression of many genes important for glucose-induced insulin release decreased progressively with increasing hyperglycemia, in parallel with a reduction of several islet transcription factors involved in beta cell development and differentiation. In contrast, genes barely expressed in sham islets (lactate dehydrogenase A and hexokinase I) were markedly increased, in parallel with an increase in the transcription factor c-Myc, a potent stimulator of cell growth. These abnormalities were accompanied by beta cell hypertrophy. Changes in gene expression were fully developed 2 weeks after pancreatectomy. Correction of blood glucose by phlorizin for the next 2 weeks normalized islet gene expression and beta cell volume without affecting plasma nonesterified fatty acid levels, strongly suggesting that hyperglycemia triggers these abnormalities. In conclusion, chronic hyperglycemia leads to beta cell hypertrophy and loss of beta cell differentiation that is correlated with changes in c-Myc and other key transcription factors. A similar change in beta cell differentiation could contribute to the profound derangement of insulin secretion in human diabetes.

Non-steroidal anti-inflammatory drug-induced apoptosis in gastric cancer cells is blocked by protein kinase C activation through inhibition of c-myc.

Apoptosis plays a major role in gastrointestinal epithelial cell turnover, ulcerogenesis and tumorigenesis. We have examined apoptosis induction by non-steroidal anti-inflammatory drugs (NSAIDs) in human gastric (AGS) cancer cells and the role of protein kinase C (PKC) and apoptosis-related oncogenes. After treatment with aspirin or indomethacin, cell growth was quantified by MTT assay, and apoptosis was determined by acridine orange staining, DNA fragmentation and flow cytometry. The mRNA and protein of p53, p21waf1/cip1 and c-myc was detected by Northern and Western blotting respectively. The influence of PKC on indomethacin-induced apoptosis was determined by co-incubation of 12-O-tetradecanoylphorbol 13-acetate (TPA). The role of c-myc was determined using its antisense oligonucleotides. The results showed that both aspirin and indomethacin inhibited cell growth and induced apoptosis of AGS cells in a dose- and time-dependent manner, without altering the cell cycle. Indomethacin increased c-myc mRNA and protein, whereas p53 and p21waf1/cip1 were unchanged. Down-regulation of c-myc by its antisense oligonucleotides reduced apoptosis induction by indomethacin. TPA could inhibit indomethacin-induced apoptosis and accumulate cells in G2/M. Overexpression of c-myc was inhibited by TPA and p21waf1/cip1 mRNA increased. In conclusion, NSAIDs induce apoptosis in gastric cancer cells which may be mediated by up-regulation of c-myc proto-oncogene. PKC activation can abrogate the effects of NSAIDs by decreasing c-myc expression. © 1999 Cancer Research Campaign

Control of human vascular smooth muscle cell proliferation by sera derived from 'experimentally stressed' individuals.

Recently we reported that individuals with high scores in standardized hostility evaluation tests, when placed in a environment, may have an association with increases in a blood-borne mitogenic substance(s) for arterial smooth muscle cells. To further investigate the molecular basis for such an association, PDS [plasma derived serum with platelet derived growth factor (PDGF) removed] from individuals showing the greatest differential pre/post-stress mitogenic activity, were tested for ability to modulate changes in the steady state of the c-myc mRNA in cultured VSMC (vascular smooth muscle cells) by Northern blot analysis. Post-stress PDS resulted in a significant increase in c-myc mRNA, when compared with pre-stress PDS of the same individual. These results give further experimental support for the notion that stress (even in the form of transient, episodic psychological challenges) may affect the cardiovascular system via rapid elicited rises in serum mitogenic activity for VSMC.

Inhibition of cell proliferation by 17beta-estradiol and heregulin beta1 in estrogen receptor negative human breast carcinoma cell lines.

Heregulin (HRG) and 17beta-estradiol (E2) interactions that modulate growth of breast cancer cell lines have recently been demonstrated. We examined the ability of heregulin beta1 (HRGbeta1) and 17beta-estradiol to modulate the biological behavior of estrogen receptor (ER) negative human breast cancer cell lines (AU-565). The proliferation of AU-565, MBA-MB231, and SKBR3 cells was additively inhibited by treatment with 17beta-estradiol (10(-6) M) and HRGbeta1 (10 ng/ml). 17-beta estradiol did not support the transcriptional activation of a reporter gene construct containing an estrogen response element transfected into AU-565 cells. This finding suggested functional endogenous ER was not present in AU-565 cells. However, the cells contained a high number of low affinity estrogen binding sites. 17beta-estradiol only slightly decreased basal tyrosine phosphorylation of ErbB-2 and ErbB-3. Estrogen and HRGbeta1 treatment resulted in an increase of c-myc mRNA. We conclude that 17beta-estradiol and HRGbeta1, in combination, potently inhibit cell proliferation of three ER negative breast carcinoma cell lines.