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F M Torti

Publications and source records attributed to F M Torti.

At least 37 records · Page 2Linked to original sources
PubMed1995

Role for NF-kappa B in the regulation of ferritin H by tumor necrosis factor-alpha.

Ferritin is a ubiquitously distributed iron-binding protein that plays a key role in cellular iron homeostasis. It is composed of two subunits, termed H (heavy or heart) and L (light or liver). In fibroblasts and other cells, the cytokine tumor necrosis factor-alpha (TNF) specifically induces synthesis of the ferritin H subunit. Using nuclear run-off assays, we demonstrate that this TNF-dependent increase in ferritin H is mediated by a selective increase in ferritin H transcription. Transfection of murine fibroblasts with chimeric genes containing the 5'-flanking region of murine ferritin H fused to the human growth hormone reporter gene reveals that the cis-acting element that mediates this response is located approximately 4.8 kilobases distal to the start site of transcription. Deletion analyses delimit the TNF-responsive region to a 40-nucleotide sequence located between nucleotides -4776 and -4736, which we term FER-2. Electrophoretic mobility shift assays and site-specific mutations indicate that this region contains two independent elements: one contains a sequence that binds a member of the NF-kappa B family of transcription factors, and a second contains a novel sequence that partially conforms to the NF-kappa B consensus sequence and may bind a different member of the NF-kappa B/Rel transcription factor family. Thus, effects of an inflammatory cytokine on ferritin are mediated by a family of transcription factors responsive to oxidative stress.

3T3 Cells↗
PubMed1995

FER-1, an enhancer of the ferritin H gene and a target of E1A-mediated transcriptional repression.

Ferritin, the major intracellular iron storage protein of eucaryotic cells, is regulated during inflammation and malignancy. We previously reported that transcription of the H subunit of ferritin (ferritin H) is negatively regulated by the adenovirus E1A oncogene in mouse NIH 3T3 fibroblasts (Y. Tsuji, E. Kwak, T. Saika, S. V. Torti, and F. M. Torti, J. Biol. Chem. 268:7270-7275, 1993). To elucidate the mechanism of transcriptional repression of the ferritin H gene by E1A, a series of deletions in the 5' flanking region of the mouse ferritin H gene were constructed, fused to the chloramphenicol acetyltransferase (CAT) gene, and transiently cotransfected into NIH 3T3 cells with an E1A expression plasmid. The results indicate that the E1A-responsive region is located approximately 4.1 kb 5' to the transcription initiation site of the ferritin H gene. Further analyses revealed that a 37-bp region, termed FER-1, is the target of E1A-mediated repression. This region also serves as an enhancer, augmenting ferritin H transcription independently of position and orientation. FER-1 was dissected into two component elements, i.e., a 22-bp dyad symmetry element and a 7-bp AP1-like sequence. Insertion of these DNA sequences into a ferritin H-CAT chimeric gene lacking an E1A-responsive region indicated that (i) the 22-bp dyad symmetry sequence by itself has no enhancer activity, (ii) the AP1-like sequence has moderate enhancer activity which is repressed by E1A, and (iii) the combination of the dyad symmetry element and the AP1-like sequence is required for maximal enhancer activity and repression by E1A. Gel retardation assays and cotransfection experiments with c-fos and c-jun expression vectors suggested that members of the Fos and Jun families bind to the AP1-like element of FER-1 and contribute to its regulation. In addition, gel retardation assays showed that E1A reduces the ability of nuclear proteins to bind to the AP1-like sequence without affecting the levels of nuclear factors that recognize the 22-bp dyad symmetry element. Taken together, these results demonstrate that FER-1 serves as both an enhancer of ferritin H transcription and a target for E1A-mediated repression.

Adenovirus E1A Proteins↗
PubMed1994

Transrectal ultrasound in the evaluation of rhabdomyosarcoma involving the prostate.

OBJECTIVE: To evaluate transrectal ultrasound as a means of diagnosis and of monitoring patients with rhabdomyosarcoma involving the prostate. PATIENTS AND METHODS: Serial transrectal ultrasonography was utilized to evaluate prostatic rhabdomyosarcoma in three patients. RESULTS: Unlike prostatic adenocarcinoma and transitional cell carcinoma involving the prostate, which are predominantly hypoechoic, the echogenicity of rhabdomyosarcoma is similar to that of the normal prostate. Transrectal ultrasound provided a simple means of monitoring prostate size and sampling tissue in these patients. CONCLUSION: Transrectal ultrasound imaging can be useful in both diagnosis and evaluation of treatment response as well as provide easy access for biopsies in patients with rhabdomyosarcoma involving the prostate.

Adolescent↗
PubMed1993

Tumor necrosis factor-induced c-myc expression in the absence of mitogenesis is associated with inhibition of adipocyte differentiation.

Tumor necrosis factor (TNF) inhibits and reverses differentiation of mouse adipogenic TA1 cells. We have found that TNF induces c-myc in a sustained manner in both preadipocytes and adipocytes; in contrast, serum induces c-myc transiently and only in preadipocytes. This TNF-mediated c-myc induction is not coupled with cell proliferation but is correlated with TNF-mediated inhibition of adipocyte differentiation. We prepared an inducible c-myc transformant of TA1 cells by transfection of the mouse c-myc gene under the control of the metallothionein-I promoter. These cells are unable to differentiate to adipocytes in the presence of Zn2+/Cd2+, and in differentiated TA1 cells, Zn2+/Cd2+ causes reduction of adipocyte-specific gene expression as does TNF. Lastly, exposure of TA1 cells to antisense c-myc oligonucleotide partially blocked the TNF-mediated reduction of adipocyte-specific gene expression. Thus, TNF-mediated c-myc expression is distinct in character from that involved in mitogenic responses but appears to play an important role in inhibition and reversal of adipocyte differentiation.

Adipocytes↗
PubMed1993

Effect of verapamil on doxorubicin cardiotoxicity: altered muscle gene expression in cultured neonatal rat cardiomyocytes.

Verapamil reverses multidrug resistance acquired by cancer cells during treatment with chemotherapeutic agents such as doxorubicin by inhibiting the function of P-glycoprotein. Verapamil has also been suggested to potentiate the cardiotoxicity of doxorubicin. We have recently demonstrated that selective inhibition of cardiac muscle gene expression is among the earliest events in doxorubicin cardiotoxicity. To explore the influence of verapamil on doxorubicin cardiotoxicity, we evaluated [14C]-doxorubicin accumulation, cardiac muscle gene expression by Northern blot analysis, and ultrastructural changes in cultured cardiomyocytes in the presence and absence of verapamil. Treatment with a combination of doxorubicin and verapamil for 24 h did not augment doxorubicin accumulation in cardiomyocytes, although substantial augmentation of doxorubicin accumulation by verapamil in cardiac fibroblasts was observed. Further, treatment with verapamil for 24 h did not augment the decrease in expression of muscle genes induced by doxorubicin (myosin light chain 2 slow, troponin I, M isoform creatine kinase). However, we found that verapamil reduced alpha-actin gene expression in a direct, doxorubicin-independent manner. Furthermore, the effect of doxorubicin plus verapamil on alpha-actin gene expression was additive over a wide range of doxorubicin and verapamil concentrations, resulting in a selective augmentation of doxorubicin-induced inhibition of gene expression for this single muscle protein gene. This was reflected in a substantial increase in cardiac myocyte damage when treatment with verapamil and doxorubicin was compared to treatment with doxorubicin alone by thin section electron microscopy. This suggests a possible mechanism by which verapamil may potentiate doxorubicin cardiotoxicity.

Animals↗
PubMed1993

Interleukin-1 beta induces interleukin-1 receptor antagonist and tumor necrosis factor binding protein in humans.

Sustained release or high levels of interleukin-1 (IL-1) and/or tumor necrosis factor (TNF), as observed after endotoxin challenge, can produce a variety of toxicities. Naturally occurring inhibitors to IL-1 and TNF, IL-1 receptor antagonist (IL-1ra) and soluble TNF receptor forms, have been detected. These proteins may function to buffer or limit the effects of these cytokines as part of a regulatory network. As part of a clinical trial of recombinant human interleukin-1 beta (rhIL-1 beta), serial plasma samples were obtained from 6 patients with metastatic melanoma treated with 30-min infusions of rhIL-1 beta for 5 consecutive days. The presence of circulating IL-1 receptor antagonist and soluble TNF binding proteins (TNF-R55-BP and TNF-R75-BP) were assessed. A maximum 86-fold increase for IL-1ra, a 7-8-fold increase for TNF-R55-BP, and a 2-3-fold increase for TNF-R75-BP were seen 2-4 h, 1 h, and 4 h, respectively, after rhIL-1 beta infusion. On each day of the treatment, the secretion of IL-1ra and release of TNF-R55-BP was observed, but there was no accumulation above baseline value for IL-1ra before each of the 5 daily infusions. Although there was a steady decrease of the 6-h postinfusion plasma levels for IL-1ra and TNF-R55-BP over the 5 treatment days, no increase of clinical side effects was noted. Two patients had measurable levels of TNF-alpha, but no correlation to TNF-binding proteins was observed. Our data show that early after rhIL-1 beta infusion the induction of IL-1ra secretion, as well as TNF-binding protein release, is observed.

Adult↗
PubMed1993

Preferential repression of the H subunit of ferritin by adenovirus E1A in NIH-3T3 mouse fibroblasts.

Ferritin is the major intracellular iron-storage protein in eucaryotic cells and plays a prominent role in maintaining intracellular iron homeostasis. We observed that transfection of NIH-3T3 mouse fibroblasts with the adenovirus E1A oncogene specifically repressed the mRNA for one of the subunits of ferritin, ferritin H. This occurred in the absence of any effect of E1A on the mRNA for the L subunit of ferritin. The repression of ferritin H was not a general feature of oncogene expression since transfection of NIH-3T3 cells with H-ras did not affect ferritin composition. Deletion of the conserved regions of E1A responsible for immortalization and transcriptional repression impaired the ability of E1A to repress ferritin H. Immunoprecipitation of ferritin in E1A transfectants demonstrated that the decrease in the ferritin H/L ratio observed at the mRNA level was also exhibited at the protein level. The E1A-dependent inhibition of ferritin H was also observed in a chimeric gene containing the ferritin H promoter ligated to the chloramphenicol acetyltransferase reporter gene, but was not observed in control genes in which chloramphenicol acetyltransferase activity was dependent on promoters derived from SV40 or the interleukin-3 gene. This suggests that E1A may repress ferritin H at the transcriptional level. These results demonstrate that the adenovirus E1A oncogene specifically modulates ferritin H expression. They also suggest that alterations in cellular iron metabolism may be among the diverse array of cellular responses induced by E1A.

3T3 Cells↗
PubMed1993

Augmentation by IL-1 alpha of tumor necrosis factor-alpha cytotoxicity in cells transfected with adenovirus E1A.

The introduction of adenovirus E1A into NIH3T3 mouse fibroblasts renders them susceptible to the cytotoxic action of TNF-alpha. We found that cells transfected with 13S E1A cDNA were not similarly rendered sensitive to IL-1 alpha; however, in cells transfected with 13S E1A cDNA, TNF cytotoxicity was augmented by treatment with IL-1. To understand the role of E1A in the cytotoxic action of these cytokines, several 13S E1A cDNA deletion mutants were constructed, transfected into NIH3T3 cells, and tested for their ability to induce TNF sensitivity in the presence and absence of IL-1. Cells transfected with mutants of 13S E1A with deletions of carboxyl-terminal amino acids 223 to 289 or 151 to 289 (conserved region 3) exhibited a minimal ability to reverse E1A-induced TNF cytotoxicity but a significant ability to reverse IL-1-mediated augmentation of TNF cytotoxicity. Cells transfected with other deletion mutants of 13S E1A, including those with an internal deletion encompassing conserved region 1 (amino acid positions 23 to 107) or a deletion including conserved regions 2 and 3 (amino acid positions 108 to 289), although initially TNF resistant, became TNF sensitive on prolonged exposure to TNF. Cells transfected with these mutants also showed a reduction in IL-1-augmented TNF cytotoxicity. The larger internal deletion of E1A at amino acid positions 23 to 150 (conserved regions 1 and 2), which includes regions important for transformation, transcriptional repression, and association with cellular proteins, resulted in the complete loss of the ability of E1A to induce TNF sensitivity even in the presence of IL-1. Immunoprecipitation experiments showed that E1A proteins in NIH3T3 cells associated with a cellular 115-kDa protein, which did not co-migrate with the RB gene product; the possible involvement of this protein in E1A-mediated TNF cytotoxicity is discussed. Taken together, these results indicate that conserved regions 1 and 2 are required for E1A-mediated TNF cytotoxicity. In contrast, maximal IL-1-augmented TNF cytotoxicity requires conserved region 3 as well as the nonconserved carboxyl-terminal region of the 13S E1A product.

3T3 Cells↗
PubMed1993

Role of protein phosphorylation in TNF-induced apoptosis: phosphatase inhibitors synergize with TNF to activate DNA fragmentation in normal as well as TNF-resistant U937 variants.

This study examined the role of protein phosphorylation in TNF induction of apoptosis in several tumor cell lines by testing the effects of agents that either stimulate or inhibit protein phosphorylation. The serine-threonine phosphatase inhibitors, okadaic acid (OKA) and calyculin A (CLA), synergistically augmented TNF-induced apoptosis in several TNF-sensitive tumor cell lines including the U937 histiocytic lymphoma, the BT-20 mammary carcinoma, and the LNCap prostatic tumor cell line. Furthermore, the phosphatase inhibitors completely reversed the TNF resistance of a variant (U9-TR) derived from U937. CLA also inhibited phosphatase activity in cell-free extracts from both U937 and U9-TR at the same concentrations (0.4-2.0 nM) that it synergized with TNF. In contrast, TNF treatment of U937 cells did not result in inhibition of phosphatase activity mediated by protein phosphatase 1 (PP1) and PP2A in cell extracts. Since the phosphatase inhibitors are known to increase the overall levels of protein phosphorylation in cells, this suggested that TNF may act by stimulating protein kinase (PK) activity. This hypothesis was supported by the results of testing a panel of relatively specific protein kinase inhibitors. TNF activation of DNA fragmentation was blocked by a potent inhibitor of myosin light chain kinase (MLCK) but was unaffected by inhibitors of cAMP or cGMP-dependent PKs. We postulate that a defect in the activation of MLCK or possibly some other as yet unknown PK may be responsible for the TNF resistance of U9-TR. Furthermore, this resistance may be circumvented by promoting protein phosphorylation with the serine-threonine-dependent phosphatase inhibitors.

Alkaloids↗
PubMed1993

Selective loss of CDC2 and CDK2 induction by tumor necrosis factor-alpha in senescent human diploid fibroblasts.

Normal human diploid fibroblasts undergo a finite number of doublings in culture. This process of senescence is accompanied by a loss in the ability to respond to proliferative stimuli and is therefore distinct from the quiescent state induced by nutrient deprivation. We have studied changes in gene expression induced in these cells following exposure to the cytokine, tumor necrosis factor-alpha (TNF). We observed that TNF induced CDC2 and CDK2 expression in early-passage quiescent WI-38 fibroblasts. However, as cells approached senescence, their ability to induce CDC2 and CDK2, as well as stimulate DNA synthesis in response to TNF, progressively declined, with minimal to absent induction in senescent cells. This occurred despite the TNF-dependent induction of such proliferation-independent genes as manganese superoxide dismutase and interleukin-6 in senescent and quiescent cells. Serum was similarly unable to induce CDC2 or CDK2 expression in senescent cells. These results demonstrate that senescent cells are selectively deficient in TNF-mediated induction of CDC2 and CDK2, genes crucial to DNA synthesis and mitosis.

CDC2 Protein Kinase↗
PubMed1993

N-acetyl-L-cysteine protects endothelial cells but not L929 tumor cells from tumor necrosis factor-alpha-mediated cytotoxicity.

The effect of N-acetyl-L-cysteine on the cytotoxicity of tumor necrosis factor-alpha was investigated in cultured bovine pulmonary artery endothelial cells and L929 mouse tumor cells. In endothelial cells, a 72-h incubation with tumor necrosis factor-alpha (100 ng/ml) reduced the number of viable cells to 27% of control. Simultaneous incubation with N-acetyl-L-cysteine (0.5-5 mmol/l) protected endothelial cells from tumor necrosis factor-alpha-mediated cytotoxicity and increased viability in a concentration-dependent fashion to 69% of control. Under the same conditions, a 72-h incubation with tumor necrosis factor-alpha (100 ng/ml) reduced the number of viable L929 tumor cells to 31% of control. However, this cytotoxic response remained unaltered in the presence of N-acetyl-L-cysteine (0.5-5 mmol/l). Similar results were obtained when using a lower concentration of tumor necrosis factor-alpha (50 ng/ml). These findings demonstrate protection from tumor necrosis factor-alpha-mediated toxicity by N-acetyl-L-cysteine in endothelial cells but not in a tumor cell line. It is concluded that N-acetyl-L-cysteine might serve as a therapeutic agent to limit the vascular toxicity of tumor necrosis factor-alpha without affecting its antineoplastic activity.

Acetylcysteine↗
PubMed1993

Cisplatin, methotrexate and vinblastine plus surgical restaging for patients with advanced transitional cell carcinoma of the urothelium.

Chemotherapy with cisplatin, methotrexate and vinblastine (CMV) is active in advanced transitional cell carcinoma of the urothelium. Aggressive surgical resection of residual disease following responses produced by CMV was incorporated into a combined modality approach. Between 1982 and 1990, 64 patients were entered into the study. Of 55 patients evaluable for response 11 (20%) had a pathological complete response, 14 (25%) achieved a complete response following resection of residual disease and 5 (9%) whose disease was not surgically restaged had a clinical complete response. The overall complete response rate was 55%. Patients with liver, lung or bone involvement had significantly decreased survival compared to patients without visceral disease (p = 0.002). With a median followup exceeding 50 months, 14 patients (22% of all patients entered into the study) were free of disease at 23 to 98+ months. There were no deaths related to treatment. CMV produced high rates of response in patients with advanced disease, including those with distant metastases. Surgical resection of residual disease following responses produced by chemotherapy proved to be feasible, without treatment related mortality, and may have prolonged survival in selected cases.

Adult↗
PubMed1993

Expression of 92-kD type IV collagenase/gelatinase (gelatinase B) in osteoarthritic cartilage and its induction in normal human articular cartilage by interleukin 1.

We report here that a 92-kD gelatinolytic metalloproteinase is expressed as protein and mRNA in human osteoarthritic cartilage, but not in normal adult articular cartilage. Western immunoblotting demonstrated that the 92-kD gelatinolytic activity corresponded to 92-kD type IV collagenase/gelatinase (gelatinase B); mRNA for gelatinase B was identified by Northern blotting. Chondrocytes from normal cartilage also exhibited mRNA for 72-kD type IV collagenase/gelatinase (gelatinase A), tissue collagenase, and stromelysin-1, and these mRNAs were increased in osteoarthritic cartilage. Regional analysis of osteoarthritic cartilage samples from four individuals revealed that gelatinase B mRNA was expressed in grossly fibrillated areas; two of four nonfibrillated cartilage samples failed to exhibit the mRNA, but did have increased levels of mRNA for other neutral metalloproteinases. IL-1 alpha treatment of normal human cartilage explants or isolated chondrocytes induced increased levels of gelatinase B and increased mRNA for tissue collagenase and stromelysin-1. Under identical conditions, mRNA levels for gelatinase A were not increased indicating that regulation of this enzyme in human articular chondrocytes is distinct from that of other metalloproteinases. Our data showing expression of gelatinase B in fibrillated cartilage suggest that it is a marker of progressive articular cartilage degradation in osteoarthritis.

Adult↗
PubMed1993

The role of iron in the cytotoxicity of tumor necrosis factor.

The effect of iron on the cytotoxicity of tumour necrosis factor-alpha (TNF, cachectin) was examined in L929 cells, which are killed by TNF at low concentrations. In L929 cells, the addition of iron (FeNTA) either prior to or concurrent with the addition of TNF markedly augmented the cytotoxicity of TNF over a wide range of TNF concentrations. Iron alone was not cytotoxic to the cells and did not inhibit protein synthesis. The iron chelator deferoxamine was able to protect against TNF cytotoxicity in L929 cells. Iron chelators also protected L929 cells from the cytotoxicity of TNF plus cycloheximide, suggesting that iron plays a role in this mode of TNF killing as well. TNF did not induce the synthesis of ferritin heavy chain in L929 cells. These experiments demonstrate that cellular iron status affects the ability of TNF to kill sensitive target cells.

Animals↗
PubMed1992

Chemotherapy of advanced transitional-cell carcinoma of the bladder.

A number of single agents and multidrug combinations are useful in the therapy of advanced transitional-cell carcinoma of the bladder. Phase II studies have identified cisplatin, Adriamycin (doxorubicin), methotrexate, and vinblastine as the most active cytotoxic agents. Combination chemotherapy based on cisplatin has shown greater efficacy than older regimens based on Adriamycin or methotrexate. Trials of regimens containing both cisplatin and methotrexate, such as those conducted by the Northern California Oncology Group using CMV (cisplatin, methotrexate, and vinblastine), have reported that a significant number of patients respond to treatment, with frequent complete responses being noted. Anthracycline-containing regimens such as M-VAC (methotrexate, vinblastine, Adriamycin, and cisplatin) have also played an important role in the therapy of advanced bladder cancer. Trials comparing cisplatin- and methotrexate-containing regimens with single-agent cisplatin or other cisplatin combinations have shown the apparent superiority of the former in terms of greater overall response rates and improved survival. However, the toxicity of such regimens can be significant, and phase III studies are under way to validate their use in the neoadjuvant setting.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1992

Transitional cell carcinoma of the prostate in patients undergoing radical cystoprostatectomy.

To assess the impact of prostatic involvement with transitional cell carcinoma we reviewed the clinical outcome of 49 patients with transitional cell carcinoma of the prostate. In addition, 115 step-sectioned cystoprostatectomy specimens removed for bladder transitional cell carcinoma were studied to determine the true incidence of secondary prostatic involvement by transitional cell carcinoma. Specimens from 300 prostates removed for prostatic adenocarcinoma also were reviewed to investigate the presence of incidental transitional cell carcinoma arising within the prostate. Transitional cell carcinoma was found in 29% of the step-sectioned specimens and in none of the radical prostatectomy specimens. The presence of prostatic invasion either into the stroma or involving prostatic ducts and acini only had no adverse effect on outcome. Lymph node status and bladder stage, and not prostatic invasion were the determining factors of survival. The presence of seminal vesicle involvement or prostatic stromal invasion appeared to predict for lymph node involvement. With a mean followup of more than 3 years 75% of our patients who had negative lymph nodes and low stage bladder lesions are alive without evidence of disease. In our series prostatic involvement by transitional cell carcinoma did not impact on survival when patients were treated aggressively with radical cystoprostatectomy.

Adult↗