Novel chemotherapeutic agent FX-9 activates NF-κB signaling and induces G1 phase arrest by activating CDKN1A in a human prostate cancer cell line.

BACKGROUND: The aminoisoquinoline FX-9 shows pro-apoptotic and antimitotic effects against lymphoblastic leukemia cells and prostate adenocarcinoma cells. In contrast, decreased cytotoxic effects against non-neoplastic blood cells, chondrocytes, and fibroblasts were observed. However, the actual FX-9 molecular mode of action is currently not fully understood. METHODS: In this study, microarray gene expression analysis comparing FX-9 exposed and unexposed prostate cancer cells (PC-3 representing castration-resistant prostate cancer), followed by pathway analysis and gene annotation to functional processes were performed. Immunocytochemistry staining was performed with selected targets. RESULTS: Expression analysis revealed 0.83% of 21,448 differential expressed genes (DEGs) after 6-h exposure of FX-9 and 0.68% DEGs after 12-h exposure thereof. Functional annotation showed that FX-9 primarily caused an activation of inflammatory response by non-canonical nuclear factor-kappa B (NF-κB) signaling. The 6-h samples showed activation of the cell cycle inhibitor CDKN1A which might be involved in the secondary response in 12-h samples. This secondary response predominantly consisted of cell cycle-related changes, with further activation of CDKN1A and inhibition of the transcription factor E2F1, including downstream target genes, resulting in G1-phase arrest. Matching our previous observations on cellular level senescence signaling pathways were also found enriched. To verify these results immunocytochemical staining of p21 Waf1/Cip1 (CDKN1A), E2F1 (E2F1), PAI-1 (SERPNE1), and NFkB2/NFkB p 100 (NFKB2) was performed. Increased expression of p21 Waf1/Cip1 and NFkB2/NFkB p 100 after 24-h exposure to FX-9 was shown. E2F1 and PAI-1 showed no increased expression. CONCLUSIONS: FX-9 induced G1-phase arrest of PC-3 cells through activation of the cell cycle inhibitor CDKN1A, which was initiated by an inflammatory response of noncanonical NF-κB signaling.

Chronic cough associated with interferon/ribavirin therapy for hepatitis C.

WHAT IS KNOWN AND OBJECTIVE: The combination of pegylated interferon and ribavirin has become standard therapy for chronic hepatitis C infection. The occurrence of chronic cough associated with this treatment regimen has been reported, but the mechanism by which cough occurs has not previously been investigated. We measured cough reflex sensitivity, during and after completion of therapy, in four patients who developed chronic cough associated with interferon/ribavirin therapy. CASE SUMMARY: Four patients without history of respiratory symptoms developed chronic cough temporally related to initiation of therapy with pegylated interferon and ribavirin for chronic hepatitis C infection. Cough resolved within 2-6 weeks after completion of a 48-week course of therapy. To measure cough reflex sensitivity, capsaicin cough challenge testing was performed 1 month prior to cessation of therapy, and 1 and 2 months after completion of treatment. In all patients, cough reflex sensitivity, as measured by C(5) , the concentration of capsaicin inducing 5 or more coughs, was significantly enhanced during treatment compared to 1 month after completion of therapy (P = 0·016). WHAT IS NEW AND CONCLUSION: Previous studies have observed that cough occurs more commonly in patients receiving the combination of interferon and ribavirin compared to interferon alone, thus implicating ribavirin as the causal agent. Our data demonstrate that it does so by reversible enhancement of cough reflex sensitivity. Clinicians should be aware of this potential treatment-related effect, so as to avoid unnecessary and costly diagnostic evaluations seeking an alternative aetiology of cough.

In vitro and in vivo association of transforming growth factor-beta 1 with hepatic fibrosis.

Despite extensive efforts, little progress has been made in identifying the factors that induce hepatic fibrosis. Transforming growth factor-beta (TGF-beta) has been shown to enhance collagen production, therefore its role in hepatic fibrosis was investigated. Treatment of cultured hepatic cells with TGF-beta 1 increased type I procollagen mRNA levels 13-fold due to post-transcriptional gene regulation. When two animal models of hepatic fibrosis, murine schistosomiasis and CCl4-treated rats, were examined, they both exhibited increased levels of TGF-beta 1 gene expression at times that somewhat preceded the increase in collagen synthesis. In contrast, in murine schistosomiasis, mRNA levels of tumor necrosis factor and interleukin-1 peaked early in the fibrogenic process. Immunohistochemical analysis showed TGF-beta 1 to be present in normal mouse liver and to be markedly increased in mice infected with schistosomiasis. TGF-beta 1 appeared in the hepatic parenchyma, primarily in hepatocytes. These findings strongly suggest a role for TGF-beta 1 in a pathophysiological state.

Molecular studies of ceruloplasmin deficiency in Wilson's disease.

Deficiency of serum ceruloplasmin is a characteristic biochemical abnormality of Wilson's disease, although the mechanism of this finding is unknown. Ceruloplasmin messenger RNA (mRNA) levels were therefore examined in five patients with Wilson's disease and five controls with other types of hepatic disease. Northern and dot blot hybridizations showed that detectable ceruloplasmin mRNA was present in all of the patients with Wilson's disease, including one patient with no detectable serum ceruloplasmin. However, the ceruloplasmin mRNA levels in the Wilson's disease patients were only 33% that of controls (P less than 0.001). In contrast, albumin mRNA levels in the Wilson's disease patients averaged 161% that of controls. In an attempt to better delineate the level of gene expression responsible for this decrease in ceruloplasmin mRNA, the nuclear run-on assay was used to analyze transcriptional rates. The amount of ceruloplasmin gene transcription in four Wilson's patients was decreased to 44% that of three controls. These results indicate that the diminished serum ceruloplasmin levels in patients with Wilson's disease are due at least in part to a decrease in ceruloplasmin gene transcription.

Gastric vascular ectases.

Gastric vascular ectases are being increasingly recognized as a significant source of acute and chronic upper gastrointestinal bleeding. Upper gastrointestinal endoscopy has not only facilitated the identification of gastric vascular ectases but has also revolutionized their therapy. This article reviews recent developments with reference to the epidemiology, diagnosis, pathogenesis, and therapy of gastric vascular ectases.

Treatment of phonological disability using the method of meaningful minimal contrast: two case studies.

The results of a treatment procedure using meaningful minimal contrasts are reported. The procedure was successful in reducing the frequency of the following processes: final consonant deletion, stopping of fricatives, and fronting of velars, which were exhibited in the speech of two children with phonological disability. Generalization of response to non-treatment words was also evidenced.

Systematic sound preference asa characteristic of phonological disability.

Systematic sound preference was described as a common phonological process seen in young children with unintelligible speech. In its classic form, sound preference occurred when an entire class of sounds was replaced by one sound. Sound preference most commonly occurred in the word-initial position and affected fricatives more frequently than any other manner category. In cases where sound preference did not affect all members of a manner of production, it affected the voiceless and/or non-labial sounds.

Effects of listener uncertainty on articulatory inconsistency.

This investigation determined the effects of listener uncertainty on articulatory inconsistency. Subjects were 15 children between three and five years of age. Each subject was tested to find a set of 45 pictures to which articulatory responses would contain sound errors on: /f,v,th,s,ts,dz/. After one week, articulatory responses to these 45 stimuli elicited by traditional picture naming techniques were compared to productions of the same words elicited in an experimental communication setting. The number of sound errors decreased significantly (p less than or equal to 0.01) in the experimental communication setting when the listener pretended to be uncertain of what the speaker said. This finding was interpreted to mean that listener uncertainty may increase the effectiveness of articulatory remediation procedures if included in treatment programs.

Adolescent Rorschach responses as a function of age at first institutionalization.

To test the hypothesis that age of first separation from a home environment is a factor in the emotional health of institutionalized boys, Rorschach protocols of 82 residents of Saint Francis Vocational School, a facility for deprived nondelinquent nonretarded youth aged 10 to 18, were examined. Seven Rorschach variables were used as dependent variables of emotional status. It was found that boys who had first been institutionized before age 10 gave more color-dominant and pure color responses (p less than .005), and more responses with pathological overtones (p less than .005), than did youth first placed at ages 10 to 13 or ages 14 to 17. Fixation was proposed as the basis for the group differences.

Amplification of the metallothionein-1 and metallothionein-2 genes in copper-resistant hepatoma cells.

The molecular basis for increased metallothionein concentrations in copper-resistant hepatoma cells was examined. The copper-resistant cell line HAC600, which is maintained in 600 microns copper, had increased steady-state mRNA levels for both the metallothionein-1 (MT-1) and the metallothionein-2 (MT-2) genes. Levels of mRNA were increased 11-fold for MT-1 and 15-fold for MT-2, with no significant change in alpha-tubulin mRNA content. HAC600NM cells, which are copper-resistant cells kept in a normal copper concentration for over 1 year, also had eight- and tenfold increases in MT-1 and MT-2 mRNA levels. Nuclear run-on assays showed that MT-1 and MT-2 gene transcription was increased nine- and eightfold in HAC600 cells and seven- and tenfold in HAC600NM cells, respectively. Southern blot analysis showed amplification of both metallothionein genes in HAC600 and HAC600NM cells. Thus the molecular basis of increased metallothionein in these hepatoma cells involved a stable gene amplification of both MT genes. The greater increase in metallothionein mRNA levels in HAC600 cells relative to the changes in transcription suggests that posttranscriptional mechanisms of gene regulation may also be acting in these cells.

Glucose, insulin and glucagon response to intravenous glucose load in patients on chronic hemodialysis.

We examined the theory that patients with chronic renal failure exhibit glucose intolerance that is not completely corrected by dialysis. I.v. glucose tolerance tests (IVGTT) were performed in 11 uremic patients who were on chronic intermittent hemodialysis therapy for a mean of 33 months, before and 24 h after dialysis. The glucose disappearance constants (K-glucose) were normal in all subjects and were not affected by dialysis. Insulin response was within normal limits, with minimal changes by dialysis. Serum glucagon was higher than normal. The early, middle and late insulin levels were the same as in the normal population. These results indicate that chronic hemodialysis therapy, together with continuous effective control of biochemical impairment, can achieve normal glucose tolerance in uremic patients.

A role for cytokines as regulators of hepatic fibrogenesis.

It is evident that hepatic fibrogenesis is a complex process involving a cascade of cytokines which interact to enhance the expression of ECM. Cytokines involved early in this cascade may serve as proinflammatory agents or as stimulators of macrophage and Ito cell activation and proliferation, while those cytokines involved later in this process may be directly fibrogenic. Furthermore, we speculate that a balance between profibrogenic and antifibrogenic cytokines normally exists but in the presence of hepatic insults, a relative super-abundance of the fibrogenic factors promotes the development of liver fibrosis. To date, most of the evidence supporting a role for cytokines in liver fibrosis has been obtained in in vitro systems or in animal models. We now need to extend these findings to man in order to determine whether a similar cascade of cytokines is important in the development of this pathologic process in man. Further delineation of these cytokines (as well as other profibrogenic soluble factors), and the mechanisms by which they act, are critical to our development of more rational forms of therapy for liver fibrosis.

Increased transforming growth factor-beta 1 gene expression in human liver disease.

We recently demonstrated that transforming growth factor-beta 1 stimulates collagen synthesis in hepatic cells in vitro, and that the synthesis of this cytokine is markedly increased in two rodent models of hepatic fibrosis. In the present study, we investigated the association of transforming growth factor-beta 1 (TFG-beta 1) gene expression in human liver disease. Sixteen patients with active liver disease had percutaneous liver biopsies performed for diagnostic purposes. Total RNA was extracted from an unused portion of each biopsy and then subjected to hybridization analysis with the following human cDNA clones: albumin, pro alpha 1 (I) collagen, and TGF-beta 1. Surgical liver biopsy specimens from two patients without hepatic disease were used as controls. When compared to controls, the patients with active liver disease had a 19% decrease in albumin, a 97% increase in type I collagen, and a 120% increase in transforming growth factor-beta 1 mRNA levels. Moreover, steady-state levels of TGF-beta 1 and procollagen mRNAs were significantly correlated. Nuclear run-on assays showed that livers from two patients with fibrosis had TGF-beta 1 transcription rates that were more than 2-fold higher than rates in control livers. These findings indicate that transforming growth factor-beta 1 gene expression is significantly enhanced in man during active liver disease.

Synchronization of cardiac autonomic neural discharge with epileptogenic activity: the lockstep phenomenon.

Autonomic dysfunction has been implicated in the sudden, unexplained deaths which account for 5-17% of mortality in persons with epilepsy. This study was designed to determine if epileptogenic activity is associated with changes in the pattern of autonomic cardiac neural discharge and the development of arrhythmias. Nine cats, anesthetized with alpha-chloralose, received pentylenetetrazol (PTZ) 10, 20, 50, 100, 200 and 2000 mg/kg, i.v. at 10 min intervals. Cardiac postganglionic sympathetic and vagal nerve discharges were correlated with the interictal spikes, brief ictal discharges (bilateral polyspikes less than 10 sec duration), and prolonged ictal discharges (polyspikes lasting greater than 10 sec). Cardiac sympathetic and vagal neural discharges were intermittently synchronized 1:1 with all 3 types of epileptogenic discharge, i.e., the lockstep phenomenon (LSP); at other times the relationship was almost 1:1 LSP was not present during control and did not always persist for the entire interval after each PTZ dose. Five of 8 cats showed LSP in the cardiac sympathetic neural discharge associated with interictal spikes induced by 10 mg/kg PTZ; 3 others exhibited LSP with interictal spikes seen subsequent to ictal discharges. The incidence of LSP was less often associated with cardiac vagal neural discharge (2 of 7 cats). Premature ventricular contractions were sometimes associated with LSP. Abnormal cardiac sympathetic and vagal neural discharge and cardiac arrhythmias were thus associated with subconvulsant (interictal) activity. Therefore, the LSP may be a factor in the mechanism of unexplained death in persons with epilepsy who exhibited no overt seizure activity at the time of demise.