TIGAR induces p53-mediated cell-cycle arrest by regulation of RB-E2F1 complex.

BACKGROUND: p53 induces cell-cycle arrest and apoptosis in cancer cells and negatively regulates glycolysis via TIGAR. Glycolysis is crucial for cancer progression although TIGAR provides protection from reactive oxygen species and apoptosis. The relation between TIGAR-mediated inhibition of glycolysis and p53 tumour-suppressor activity is unknown. METHODS: RT-PCR, western blot, luciferase and chromatin immunoprecipitation assays were used to study TIGAR gene regulation. Co-IPP was used to determine the role of TIGAR protein in regulating the protein-protein interaction between retinoblastoma (RB) and E2F1. MCF-7 tumour xenografts were utilised to study the role of TIGAR in tumour regression. RESULTS: Our study shows that TIGAR promotes p21-independent, p53-mediated G1-phase arrest in cancer cells. p53 activates the TIGAR promoter only in cells exposed to repairable doses of stress. TIGAR regulates the expression of genes involved in cell-cycle progression; suppresses synthesis of CDK-2, CDK-4, CDK-6, Cyclin D, Cyclin E and promotes de-phosphorylation of RB protein. RB de-phosphorylation stabilises the complex between RB and E2F1 thus inhibiting the entry of cell cycle from G1 phase to S phase. CONCLUSION: TIGAR mediates de-phosphorylation of RB and stabilisation of RB-E2F1 complex thus delaying the entry of cells in S phase of the cell cycle. Thus, TIGAR inhibits proliferation of cancer cells and increases drug-mediated tumour regression by promoting p53-mediated cell-cycle arrest.

Stool caproic acid for screening of Clostridium difficile.

Clostridium difficile is the prime etiologic agent in the production of pseudomembranous colitis by its powerful cytotoxin. The most common test for the toxin is a tissue culture method with neutralization of cytopathic effect by a C. difficile antiserum. This method is expensive and requires a minimum of 72 hours before results can be obtained. Attempts to create a rapid method, counterimmunoelectrophoresis, enzyme-linked immunosorbent, latex agglutination, and fluorescent antibody test are fraught with many problems. This report describes a rapid method for the identification of C. difficile, using gas-liquid chromatography (GLC) for the demonstration of caproic acid, a product of the organisms fatty acid metabolism.

Evaluation of staining methods for identifying Campylobacter pylori.

Campylobacter pylori has been implicated in the pathogenesis of peptide ulcer disease. The rapid identification of this organism may depend upon histologic diagnosis, because culture methods are complex and require a minimum of seven days in order to identify a negative specimen. The purpose of this study was to determine which stain used to identify this organism was the most cost-effective and easiest to perform and interpret on a routine basis. Sixty-one consecutive gastric antral biopsies were stained with hematoxylin and eosin, Giemsa, Brown-Brenn, and Warthin-Starry, with 23 of the cases stained by Brown-Hopps. Of the stains tested, the Wright-Giemsa was the easiest to perform. The organisms on the Wright-Giemsa showed a smooth, uniform purple color, whereas the Warthin-Starry gave the organism a granular appearance that at times could be confused for silver precipitate. Both the Wright-Giemsa and Brown-Hopps stain had the highest degree of identification of the organism (defined by percent positivity). The routine use of the Wright-Giemsa stain for identification of C. pylori in antral biopsies is recommended.

Influence of streptozotocin (STZ)-induced diabetes, dextrose diuresis and acetone on cisplatin nephrotoxicity in Fischer 344 (F344) rats.

The following studies examined the impact of the diabetic state on cisplatin nephrotoxicity. This study also investigated the potential mechanisms for diabetes mediated reduction of cisplatin toxicity. A diabetic state was induced in male Fischer 344 (F344) rats after intraperitoneal (i.p.) injection of 27-35 mg/kg STZ. Cisplatin (5 mg/kg, i.p.) nephrotoxicity was examined in normoglycemic and diabetic rats after 48 and 96 h. Cisplatin was nephrotoxic within 96 h to normoglycemic animals as indicated by an increased kidney weight, marked elevations in serum BUN levels as well as significant P less than 0.05) decreases in renal cortical slice accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA). Cisplatin failed to depress renal cortical slice accumulation of PAH and TEA in the diabetic rats. Cisplatin was also less effective in increasing BUN levels or kidney weight in diabetic rats. Further studies investigated the impact of glycosuric diuresis and ketone bodies on cisplatin nephrotoxicity. Dextrose diuresis of normoglycemic rats failed to reduce the effect of cisplatin on BUN levels, kidney weight and renal cortical slice uptake of PAH and TEA. Acetone pretreatment of normoglycemic rats also did not reduce cisplatin nephrotoxicity. These results indicate: (1) cisplatin nephrotoxicity is attenuated in the experimental diabetic state, (2) diabetes does not reduce cisplatin nephrotoxicity through glycosuric diuresis and (3) ketone body accumulation does not modulate cisplatin nephrotoxicity.

Modification of P450 activity and its effect on 1,2-dichlorobenzene toxicity in Fischer 344 rats.

This study examined the contribution of biotransformation by the mixed function oxidase system on hepatic and renal toxicity of 1,2-dichlorobenzene (1,2-DCB). Male Fischer 344 (F344) rats (190-250 g) were pretreated with phenobarbital (PB), beta-naphthoflavone (BNF), pyridine (PYR), piperonyl butoxide (PiBx) or vehicle prior to the administration of 2 or 3 mmol/kg of 1,2-DCB. Pair-fed control animals were treated with corn oil, (1 ml/kg). Plasma alanine amino-transaminase (ALT/GPT) was increased in a dose-dependent manner by 1,2-DCB. Pretreatment with PB, BNF or PB pretreatment prior to 1,2-DCB administration increased hepatic toxicity within 24 h. Toxicity was characterized by increased ALT/GPT activity and increased liver weight. Acute administration of 1,2-DCB produced renal alterations within 24 h. Renal toxicity was characterized by altered blood urea nitrogen (BUN) concentration and decreased renal cortical slice accumulation of p-aminohippurate (PAH) 24 h after injection of 3 mmol/kg 1,2-DCB. Pretreatment with PB, BNF or PYR increased the renal toxicity of 2 and 3 mmol/kg 1,2-DCB. Conversely, pretreatment with PiBx to inhibit P450 activity slightly decreased the hepatic and renal toxicity of 1,2-DCB. These results establish that the kidney was a target organ for 1,2-DCB toxicity and that the proximal tubule was a site of damage. Additionally, these studies indicate induction of P450 isozymes increased the hepatic and renal toxicity of 1,2-DCB. Further studies are needed to examine the specific role of P450 in generation of toxicity.

Renal accumulation and urinary excretion of cisplatin in diabetic rats.

Previous work has demonstrated that cisplatin nephrotoxicity was attenuated in streptozotocin (STZ)-induced diabetic rats. The following studies investigated the hypothesis that renal cisplatin accumulation was reduced in diabetic rats. Male Fischer 344 (F344) rats were injected with 32 mg/kg STZ (i.p.) or citrate buffer. Renal platinum (Pt) accumulation was quantitated 0-96 h after the administration of 5 mg/kg cisplatin (i.p.) to normoglycemic and diabetic rats (greater than or equal to 4/group). Total renal Pt accumulation was decreased (P less than 0.05) in the diabetic rats, when compared to the normoglycemic group, 6-48 h after cisplatin injection. Further studies were also conducted to examine if urinary cisplatin excretion was enhanced in diabetic relative to normoglycemic groups. Urinary Pt excretion was quantitated 0-96 h following cisplatin (5 mg/kg, i.p.) administration. Pt excretion was increased in the diabetic group relative to the normoglycemics when comparisons were made on the basis of Pt excreted per hour or cumulative Pt excretion. Differences were also detected in urinary Pt concentration. The diabetic group had a lower urinary concentration of the metal 12-96 h after cisplatin injection. These findings suggest that the reduction in nephrotoxicity in diabetic rats may be at least partially due to decreased renal accumulation as well as altered renal excretion.

Cephaloridine nephrotoxicity in streptozotocin induced diabetic Fischer 344 (F344) rats.

The purpose of this study was to determine if cephaloridine nephrotoxicity is attenuated in streptozotocin (STZ)-induced diabetic rats. Fischer 344 (F344) rats (205-250 g) were given a single injection (i.p.) of STZ (27-35 mg/kg) or citrate buffer. The nephrotoxicity of (750 mg/kg) cephaloridine (i.p.) was then compared with normoglycemic and 14-day diabetic rats. Increased blood urea nitrogen (BUN) levels as well as diminished renal cortical slice accumulation of tetraethylammonium (TEA) and lactate-stimulated p-aminohippurate (PAH) were measured (P less than 0.05) in normoglycemic rats 48 h after cephaloridine administration. Cephaloridine failed to alter BUN levels and organic ion accumulation in diabetic rats. Diabetes did not totally protect against cephaloridine toxicity since kidney weights were elevated in normoglycemic and diabetic rats 48 h after administration of 750 mg/kg cephaloridine. A series of experiments also measured BUN levels, kidney weight and renal cortical slice uptake of PAH and TEA 24, 48 and 72 h after (1500 mg/kg) cephaloridine administration. Cephaloridine increased (P less than 0.05) kidney wt and decreased PAH and TEA uptake (P less than 0.05) in the normoglycemic group at 24-72 h. No change in kidney wt, PAH or TEA uptake was observed in the diabetic rats. These data indicate diabetes reduces cephaloridine nephrotoxicity.

Acute renal and hepatic toxicity of 2-haloanilines in Fischer 344 rats.

Aniline and its halogenated derivatives are widely used as chemical intermediates. The purpose of this study was to determine the hepatotoxic and nephrotoxic potential of the 2-haloanilines. Male Fischer 344 rats (n > or = 4) were injected (i.p.) with 1.0 or 1.25 mmol/kg of: aniline (A), 2-fluoroaniline (2-FA), 2-chloroaniline (2-ClA), 2-bromoaniline (2-BrA), 2-iodoaniline (2-IA) or vehicle (0.9% saline, 2.5 ml/kg). All compounds were injected as hydrochloride salts. Renal and hepatic function was monitored 24 h after treatment. All of the 2-haloanilines induced oliguria, diminished kidney weight, tubular casts and decreased renal cortical slice accumulation of organic anions. Blood urea nitrogen (BUN) levels were increased (P < 0.05) by treatment with 1.0 or 1.25 mmol/kg of 2-FA, 2-ClA or 2-BrA. Hepatic alterations were also observed and characterized by elevated plasma ALT/GPT activity and altered morphology in the centrilobular region. The nephrotoxic and hepatotoxic potentials were similar among the 2-haloanilines but aniline was less toxic than its 2-halo derivatives. These results demonstrated that halogen substitution at the 2-position of aniline increased hepatic and renal toxicity. However, the severity of toxicity was not influenced by the nature of the halogen substituent.

Urine screening: an alternative to urine cultures.

Urine cultures represent one of the most commonly submitted specimens to a microbiology laboratory. In an attempt to manage effectively this type of specimen, Bac-T-Screen (BTS), nitrate and leukoestrase test were performed on all clean catch urine specimens sent for culture. Three hundred and four specimens were screened by these methods. The BTS, by itself, had the highest sensitivity, and specificity (92 percent and 81 percent, respectively) and the lowest false negative rate (9.8 percent) of the three tests. A combination BTS/leukoestrase test increased specificity (99 percent), but the sensitivity was only 73 percent.

Isolation of genital mycoplasmas and Chlamydia trachomatis in stillborn and neonatal autopsy material.

Chlamydia trachomatis and the genital mycoplasmas are significantly prevalent in sexually active women. How these organisms may affect the outcome of pregnancy and the neonate was the principal thrust of this investigation. Placenta, liver, and lung tissue were cultured from Mycoplasma hominis, Ureaplasma urealyticum, Chlamydia trachomatis, and aerobic as well as anaerobic bacteria in 432 stillborn and neonatal autopsies. Genital mycoplasmas were isolated from 36 cases (8.3%). Acute chorioamnionitis and funisitis were present significantly more often in cases with genital mycoplasma than in those without these organisms. Isolation of genital mycoplasmas was not associated with an increased incidence of intrauterine fetal death, villitis, hyaline membrane disease, congenital anomalies, or polymorphonuclear leukocytes in alveolar spaces. Chlamydia trachomatis was not found in any of the sites sampled.

Histologic manifestations of perinatal genital mycoplasmal infection.

Genital mycoplasmas are controversial gynecologic and obstetric pathogens; their role in perinatal morbidity and mortality is ill defined. This retrospective study was conducted to identify autopsy manifestations of perinatal genital mycoplasmal infection. Bacterial and mycoplasmal cultures were obtained from multiple organ sites, along with detailed clinical, autopsy, and histologic information. All materials were screened by two pathologists. Twenty-nine cases were included in the study. In 17 only a genital mycoplasma was isolated, and in 12 cases a genital mycoplasma plus another bacterial pathogen were identified. Significant histologic findings included subpericardial and pleural hemorrhages, polymorphonuclear leukocytes in alveolar spaces, hemorrhage, and tubulocytic changes in the adrenal glands. The placentas revealed villous edema, deciduitis, and funicitis. The changes identified as significant suggest that mycoplasmas either cause hypoxia or promote histologic changes that mimick hypoxia.

Chorioamnionitis: a study of organisms isolated in perinatal autopsies.

The purpose of this study was to determine the validity of postmortem cultures in perinatal autopsies; in particular, those cases where there is gross or histologic evidence of chorioamnionitis. Lung, liver, blood, and placental cultures were obtained from 159 neonatal autopsies with histologic evidence of chorioamnionitis at Magee-Women's Hospital between January 1980 and July 1985. The criterion for chorioamnionitis was a polymorphonuclear leukocytic infiltrate in the placental membranes. The neonates studied were 63 percent white and 36 percent black; the male to female ratio was 83 to 76. Intrauterine fetal death occurred in 43 percent of the cases. The mean gestational age was 24 weeks. Premature labor occurred in 64 percent of the cases, and 70 percent of the cases presented with premature rupture of fetal membranes. Congenital pneumonia (defined by the presence of polymorphonuclear leukocytes in the alveolar spaces) was present in 45 percent of cases. The lung was the most frequent site cultured; the four most frequently isolated organisms were: Staphylococcus epidermidis--18 percent; Beta Streptococcus Group B--13 percent; E. coli--nine percent; Ureaplasma urealyticum--nine percent. Negative cultures from multiple sites occurred in seven percent of cases. The results of this investigation indicate that multi-organ cultures help in defining the role of a particular bacteria as a pathogen, and that Staphylococcus epidermidis may be a true fetal pathogen under certain conditions. It is recommended that cultures for Chlamydia trachomatis not be done on fetal tissues.

Histologic characteristics of Campylobacter pylori (Helicobacter pylori) mediated gastritis.

One hundred-nineteen specimens were reviewed to determine whether or not there were histologic changes specific for Campylobacter pylori (CP), (Helicobacter pylori) mediated gastritis. Hematoxylin and eosin (H&E), Brown-Hopp, and Wright-Giemsa stained sections were examined independently by two pathologists for (a) the presence of acute cryptitis, (b) percent and degree of crypt involvement, and (c) spectrum of inflammatory cells within the lamina propriae. The amount of mucus was quantified on the Periodic Acid Schift (PAS)-Alcian Blue stain sections. Changes in the character of the mucus were noted by using both the PAS-Alcian Blue and the High Iron Diamine-Alcian Blue. A positive specimen for Campylobacter pylori (CP+), (Helicobacter pylori) was defined as one in which curved or spiral shaped microbes were identified on Wright-Giemsa and Brown-Hopp stain. Seventy-eight specimens were CP+ and 41 CP-. Statistically significant histologic findings included the extent and degree of superficial cryptitis and the preponderance of plasma cells in CP+ cases. These findings confirm aspects seen in an animal model and suggest that there is an histologic pattern consistent with C. pylori (Helicobacter pylori) mediated gastritis.

Extramedullary plasmacytoma of the soft palate.

A 59-year-old white male presented with symptoms of an upper respiratory infection and the sensation of nasal fullness or obstruction. There were no constitutional symptoms or history of previous oropharyngeal neoplasms. Examination revealed a fleshy tan-pink 2 cm. x 1 cm. pedunculated lesion at the base of the uvula posteriorly, extending into the posterior soft palate. Indirect laryngoscopy confirmed extension of the lesion base onto the posterior soft palate. There was no palpable cervical adenopathy. CT scan of the sinuses and neck revealed no abnormality except the soft tissue mass in the area of the uvula and soft palate. Excisional biopsy of the lesion revealed a plasmacytoma which produced a monoclonal lambda chain immunoglobulin. Hemoglobin was 17.2 g/d. Serum electrophoresis and immunophoresis, Bence-Jones protein, bone marrow, and bone scan were normal. Twenty-six months later, a 1.5 cm. pedunculated lesion was noted at the site of original tumor, which was demonstrated to be recurrent plasmacytoma on excisional biopsy. No other lesions were identified on direct laryngoscopy and skeletal survey was normal. Repeat laboratory studies were normal except for a slight hypergammaglobulinemia (total protein 6.4 g/d., albumin 51.5 percent, alpha 1 globulin 4.0 percent, alpha 2 globulin 11.4 percent, beta globulin 12.8 percent, and gamma globulin 20.2 percent). The patient refused further treatment and has had no further recurrences at one year.

Giant basal cell carcinoma of the back.

Basal cell carcinoma accounts for 65-75 per cent of all skin carcinomas. We describe the case of a basal cell carcinoma of the back that was allowed to grow to a giant size. Even when this size, basal cell carcinomas rarely metastasize.

FAT1 acts as an upstream regulator of oncogenic and inflammatory pathways, via PDCD4, in glioma cells.

Glioblastoma multiforme (GBM) is the most aggressive and the commonest primary brain tumor with a tendency for local invasiveness. The pathways of neoplasia, invasion and inflammation are inextricably linked in cancer and aberrations in several regulatory pathways for these processes have been identified. Here we have studied the FAT1 (Homo sapiens FAT tumor-suppressor homolog 1 (Drosophila)) gene to identify its role in the tumorigenecity of the gliomas. The expression of FAT1 was found to be high in grade IV glioma cell lines (U87MG, A172, U373MG and T98G) but low in grade III glioma cell lines (GOS3 and SW1088). Two cell lines (U87MG and A172) with high FAT1 expression were chosen for in vitro FAT1-knockdown studies. FAT1 knockdown by small interfering RNA resulted in decreased migration and invasion of both the cell lines along with increased expression of the tumor-suppressor gene programmed cell death 4 (PDCD4). Increased PDCD4 expression led to the attenuation of activator protein-1 (AP- 1) transcription by inhibiting c-Jun phosphorylation and resulted in concomitant decrease in the expression of AP-1-target genes like MMP3, VEGF-C and PLAU, the pro-inflammatory regulator COX-2 and cytokines IL1b and IL-6. Conversely, simultaneous silencing of PDCD4 and FAT1 in these cells significantly enhanced AP-1 activity and expression of its target genes, resulting in increase in mediators of inflammation and in enhanced migratory and invasive properties of the cells. We also observed a negative correlation between the expression of FAT1 and PDCD4 (P = 0.0145), a positive correlation between the expression of FAT1 and COX-2 (P = 0.048) and a similar positive trend between FAT1 and IL-6 expression in 35 primary human GBM samples studied. Taken together, this study identifies a novel signaling mechanism mediated by FAT1 in regulating the activity of PDCD4 and thereby the key transcription factor AP-1, which then affects known mediators of neoplasia and inflammation.

Re-oxygenation causes hypoxic tumor regression through restoration of p53 wild-type conformation and post-translational modifications.

Hypoxic tumors are resistant to conventional therapies through indirect mechanisms such as the selection of resistant phenotype under chronic hypoxia. Hyperbaric oxygen (HBO) therapy has been shown to increase oxygen level and induce apoptosis in hypoxic tumor. However, it could produce significant adverse effects including oxygen toxic seizures and severe radiation tissue injury due to high pressure. We have shown that repeated oxygenation at 30% O(2) (1 atmospheres absolute) results in significant regression of MCF-7 tumor xenografts without any adverse effect. In MCF-7 cells, re-oxygenation showed an eightfold increase in cellular apoptosis. Both in hypoxic tumor and in hypoxic cells, that exclusively favor p53 to exist in mutant conformation, re-oxygenation restores p53 wild-type conformation. The oxygen-mediated rescue of mutant p53 followed by its trans-activation is responsible for the induction of p53-downstream apoptotic, cell-cycle arrest and DNA-repair genes. Further, p53 trans-activation may thus be due to its post-translational modifications as a result of re-oxygenation. We have thus concluded that oxygen therapy without pressure, as opposed to HBO therapy, may be ideal for hypoxic tumor regression, which functions through oxygen-mediated rescue of mutant p53 followed by induction of apoptosis.

Egg yolk emulsion agar, a new medium for the cultivation of Helicobacter pylori.

We developed a new agar, egg yolk emulsion (EYE) agar, for cultivation of Helicobacter pylori. EYE agar contains Columbia agar base (Oxoid), 10% EYE (Oxoid), 1% IsoVitaleX (BBL), and 40 mg of Triphenyleteraxolium chloride (Sigma) per liter. We compared EYE agar with the following agars: (i) brain heart infusion agar-7% horse blood-1% IsoVitaleX (GDW agar; C. S. Goodwin, E. D. Blincow, J. R. Warren, T. E. Waters, C. R. Sanderson, and L. Easton, J. Clin. Pathol. 38:1127-1131, 1985), (ii) brain heart infusion agar-10% horse serum-0.2% charcoal-1% yeast extract-40 mg of triphenyltetrazolium chloride per liter (GLU agar; Y. Glupczynski, M. Labbe, and F. Thiabaumont, p. 3-6, in F. Megraud and H. Lamouliatte, ed., Gastroduodenal Pathology and Campylobacter pylori, 1989), (iii) Columbia agar with 7% lysed horse blood (D&M agar; J. C. Dent and C. A. M. McNulty, Eur. J. Clin. Microbiol. Infect. Dis. 7:555-558, 1988), and (iv) brain heart infusion agar-10% EYE-1% IsoVitaleX (BHIE agar). H. pylori CFU counts, expressed as average percentages of maximum growth, were as follows: EYE agar, 96; GDW agar. 76; BHIE agar, 57; D&M agar, 52; and GLU agar, 23. Colony counts for EYE agar were significantly higher than for GDW agar (P = 0.027), BHIE agar (P = 0.005), D&M agar (P = 0.0001), and GLU agar (P less than 0.0001). EYE agar also had higher CFU counts than two commercial chocolate media; the EYE agar count was 80%, versus 33% for BBL chocolate medium and 63% for Remel chocolate medium.

Attenuation of cisplatin nephrotoxicity by streptozotocin-induced diabetes.

The therapeutic use of cisplatin is associated with acute renal failure. The purpose of this study was to determine (a) if streptozotocin (STZ) was toxic to renal proximal tubules and (b) the nephrotoxicity of cisplatin in STZ-diabetic rats. Male Sprague-Dawley rats were injected with STZ (55 mg/kg, ip) to induce a diabetic state. BUN and renal cortical slice uptake of p-aminohippurate (PAH) and tetraethylammonium (TEA) were not altered, relative to normoglycemic rats, 3, 16, and 28 days following STZ treatment. These results indicate that STZ is not toxic to renal proximal tubules. Cisplatin nephrotoxicity studies were then conducted in STZ-diabetic and normoglycemic rats. Cisplatin nephrotoxicity was also evaluated in diabetic rats pretreated for 8 days with insulin. Diabetic and normoglycemic rats were administered 5 mg/kg cisplatin or water (ip). Increased kidney weight, BUN levels, glucosuria, and proteinuria were measured in normoglycemic rats 4 days after cisplatin administration. Renal cortical TEA and lactate-stimulated PAH uptake (p less than 0.05) were diminished in the normoglycemic rats 4 days after cisplatin injection. No change in kidney weight, BUN levels, or renal cortical slice accumulation of PAH and TEA was observed in diabetic rats treated with cisplatin. However, cisplatin administration to diabetic rats pretreated with insulin resulted in increased mortality, proteinuria, glucosuria and elevated kidney weight. These results indicate that the diabetic state attenuates cisplatin nephrotoxicity. Additionally, these results indicate that diabetes attenuation of cisplatin nephrotoxicity is dependent on the severity of the diabetic state.