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Biomedical subjects

S E Dunn

Publications and source records attributed to S E Dunn.

At least 19 recordsLinked to original sources
PubMed2001

Nerve activity-dependent modulation of calcineurin signaling in adult fast and slow skeletal muscle fibers.

This study tested the hypothesis that calcineurin signaling is modulated in skeletal muscle cells by fluctuations in nerve-mediated activity. We show that dephosphorylation of NFATc1, MEF2A, and MEF2D transcription factors by calcineurin in all muscle types is dependent on nerve activity and positively correlated with muscle usage under normal weightbearing conditions. With increased nerve-mediated activity, calcineurin dephosphorylation of these targets was found to be potentiated in a way that paralleled the higher muscle activation profiles associated with functional overload or nerve electrical stimulation conditions. We also establish that muscle activity must be sustained above native levels for calcineurin-dependent dephosphorylation of MEF2A and MEF2D to be transduced into an increase in MEF2 transcriptional function, suggesting that calcineurin cooperates with other activity-linked events to signal via these proteins. Finally, examination of individual fiber responses to overload and nerve electrical stimulation revealed that calcineurin-MEF2 signaling occurs in all fiber types but most readily in fibers that are normally least active (i.e. those expressing IIx and IIb myosin heavy chain (MHC)), suggesting that signaling via this phosphatase is also dependent upon the activation history of the muscle cell.

Animals↗
PubMed2001

Up-regulation of urokinase-type plasminogen activator by insulin-like growth factor-I depends upon phosphatidylinositol-3 kinase and mitogen-activated protein kinase kinase.

Elevated levels of urokinase plasminogen activator-1 (uPA) and the insulin-like growth factor-I receptor (IGF-IR) are associated with breast cancer recurrence and decreased survival. It is possible that activation of IGF-IR and elevations in uPA are mechanistically linked. Our laboratory recently showed that insulin-like growth factor-I (IGF-I) induces uPA protein and mRNA in the breast cancer cell line MDA-MB-231. We also found that IGF-IR and uPA were commonly overexpressed in primary breast cancers. In this study, we investigated the signal transduction pathway through which IGF-I regulates uPA. Phosphatidylinositol 3-kinase, mitogen-activated protein kinase kinase, and p70 kinase were inhibited with LY294002, PD98059, and rapamycin, respectively. Induction of uPA protein by IGF-I was partially inhibited by LY294002 (60% inhibition) or PD98059 (30% inhibition) but not by rapamycin. The production of uPA protein induced by IGF-I was blocked up to 90% by the tyrosine kinase inhibitor herbimycin A. Furthermore, herbimycin A suppressed the phosphorylation of AKT and Erk1/2. Next, we tested the impact of the signal transduction inhibitors on uPA gene expression. Both LY294002 and PD98059 were required to completely inhibit uPA mRNA expression, whereas each drug alone resulted in approximately 50% reduction in uPA expression. Next, using a minimal uPA-luciferase promoter construct containing the binding sites for the AP-1 and Ets transcription factors, we observed that IGF-I stimulated the uPA promoter via these sites. Furthermore, both Ly294002 and PD98059 were necessary to block IGF-I-stimulated uPA-Luc activity. In summary, we conclude that IGF-I requires both phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase-dependent pathways to optimally induce uPA expression. These findings suggest that the development of drugs targeting these pathways may benefit breast cancer patients at a high risk of recurrence, such as those who have primary tumors overexpressing IGF-IR and uPA.

Benzoquinones↗
PubMed2000

Matching of calcineurin activity to upstream effectors is critical for skeletal muscle fiber growth.

Calcineurin-dependent pathways have been implicated in the hypertrophic response of skeletal muscle to functional overload (OV) (Dunn, S.E., J.L. Burns, and R.N. Michel. 1999. J. Biol. Chem. 274:21908-21912). Here we show that skeletal muscles overexpressing an activated form of calcineurin (CnA*) exhibit a phenotype indistinguishable from wild-type counterparts under normal weightbearing conditions and respond to OV with a similar doubling in cell size and slow fiber number. These adaptations occurred despite the fact that CnA* muscles displayed threefold higher calcineurin activity and enhanced dephosphorylation of the calcineurin targets NFATc1, MEF2A, and MEF2D. Moreover, when calcineurin signaling is compromised with cyclosporin A, muscles from OV wild-type mice display a lower molecular weight form of CnA, originally detected in failing hearts, whereas CnA* muscles are spared this manifestation. We also show that OV-induced growth and type transformations are prevented in muscle fibers of transgenic mice overexpressing a peptide that inhibits calmodulin from signaling to target enzymes. Taken together, these findings provide evidence that both calcineurin and its activity-linked upstream signaling elements are crucial for muscle adaptations to OV and that, unless significantly compromised, endogenous levels of this enzyme can accommodate large fluctuations in upstream calcium-dependent signaling events.

Adaptation, Physiological↗
PubMed2000

The insulin-like growth factor-1 elevates urokinase-type plasminogen activator-1 in human breast cancer cells: a new avenue for breast cancer therapy.

Tumor recurrence is a common problem in the treatment of breast cancer. In breast cancer, the expression of high protein levels of the insulin-like growth factor-1 receptor (IGF-1R) and urokinase-type plasminogen activator-1 (uPA) is strongly associated with breast cancer recurrence and decreased survival. The expression of uPA by tumors is thought to not only stimulate tumor invasion but also facilitate angiogenesis. In this study, our goal was to address whether IGF-1R could influence the expression of the extracell ular matrix proteases, matrix metalloproteinase (MMP), or uPA thus allowing a selective advantage for tumor invasion and concomitant neovascularization. Initially, we determined whether or not insulin-like growth factor (IGF)-1 regulated the production MMP or uPA in the human breast cancer MDA-MB-231 cells. There was no increase in MMP activity when the cells were treated with IGF-1 (10 ng/mL) for 24 h. In contrast, uPA mRNA and protein were induced in a time-dependent manner. Furthermore, clones expressi ng a dominant negative inhibitor of IGF-1R termed 486stop had less uPA mRNA, and the clones were less invasive through Matrigel. Taken together, these data illustrate that IGF-1R stimulates uPA production. Hence, these two prognostic indicators may be interrelated, suggesting they may function in a synergistic manner to facilitate local tumor invasion as well as angiogenesis. Our data suggest that disruption of IGF-1 signaling in breast cancer may lead to breast cancer prevention and intervention by decreasing uPA expression.

Blotting, Northern↗
PubMed1999

Calcineurin is required for skeletal muscle hypertrophy.

Molecular signaling pathways linking increases in skeletal muscle usage to alterations in muscle size have not been identified. In the present study, we tested the hypothesis that calcineurin, a calcium-regulated phosphatase recently implicated in the signaling of some forms of cardiomyopathic growth, is required to induce skeletal muscle hypertrophy and muscle fiber type conversions associated with functional overload in vivo. Administration of the specific calcineurin inhibitors cyclosporin (CsA) or FK506 to mice, for which the fast plantaris muscle was overloaded for 1-4 weeks, prevented the rapid doubling of mass and individual fiber size and the 4-20-fold increase in the number of slow fibers that characterize this condition. CsA treatment influenced the expression of muscle myofibrillar protein genes in a way reflective of fiber phenotype transformations but only in the long term of the overload condition, suggesting that the control of this growth response by calcineurin is not limited to the transcriptional activation of these muscle-specific genes. Clinically, these results provide insight to the post-surgical muscle wasting and weakness observed in recovering transplant recipients administered therapeutic dosages of these immunosuppressants.

Animals↗
PubMed1999

Differential sensitivity of myosin-heavy-chain-typed fibers to distinct aggregates of nerve-mediated activation.

We studied the regulatory effects of nerve-mediated activity on the early expression of embryonic and adult myosin heavy chains (MHC) within inactive though still innervated rat plantaris and soleus muscle fibers. To this end, we stimulated motor nerves that were quiescent following treatment with tetrodotoxin (TTX) with paradigms designed to partition the influence of neural activation frequency and assessed the selective expression and accumulation of MHCs within muscle fibers using an array of specific antibodies. We show rapid de novo expression of IIx MHC within select soleus fibers in response to high-frequency activation for more than 0.01% of daily time. High-frequency aggregates were also the most effective in preventing the TTX-induced reexpression of embryonic MHCs within specific fibers. Only configurations that included high-frequency trains for more than 0.01% of daily time or combined with 10 Hz stimulation preserved the size of select fibers, used as a measure of the net cellular content of MHC. The effectiveness of this preservation varied according to the muscle type and MHC expressed, and, in a subset of fibers, was influenced by contractile loading status. Our results demonstrate that distinct subsets of MHC-typed fibers are differentially sensitive to the neural activation cues mediating the cellular expression of these proteins.

Aging↗
PubMed1999

Roles for insulin-like growth factor-1 in mediating the anti-carcinogenic effects of caloric restriction.

This paper focuses on the role of insulin-like growth factor-1 (IGF-1) and its associated regulatory apparatus as a key endocrine, autocrine, and paracrine signalling system involved in mediating the anti-carcinogenic activity of dietary restriction. Literature is reviewed showing that the inhibitory action of dietary restriction on carcinogenesis is global and pervasive--it is effective in several laboratory species, for a variety of tumor types, and for both spontaneous tumors and tumors caused by different types of tumor-inducing agents. Evidence is presented showing the IGF-1 pathway responds appropriately to nutritional interventions including diet restriction. Recent evidence points to an obligatory role for the IGF-1 receptor in the establishment and maintenance of the transformed phenotype and reveals that IGF-1 in concert with insulin-like binding protein 3 and p53 is involved in autocrine/paracrine growth signaling pathways as adaptive responses to environmental stimuli. Considered together these works show that the IGF-1 pathway is uniquely poised to influence cellular transformation leading to the malignant phenotype by modulating the balance of cellular proliferation and cell death (apoptosis) in precancerous and cancerous cells and by influencing metastasis of nascent tumors. We evaluated these hypotheses directly using animal models of mononuclear cell leukemia, bladder transitional cell carcinogenesis, and breast cancer. Our studies demonstrate that manipulation of IGF-1 level through dietary intervention influences tumor growth and metastasis. Upregulation of this pathway demonstrated that increased IGF-1 stimulates tumor proliferation, progression and metastasis. Conversely, downregulation of this pathway in vivo as a consequence of dietary restriction results in antitumorigenic activity. We found that the functional disruption of IGF-1R markedly influences breast cancer metastasis in nude mice by suppressing cellular adhesion, invasion, and metastasis of breast cancer cells to the lung, lymph nodes, and lymph vessels. Epidemiological observations and clinical oncology results support the involvement of IGF-1 in carcinogenesis and anticarcinogenesis. This leads to the hypothesis that factors such as IGF-1 which regulate body size and composition may be related to human cancer incidence or prognosis. Additional understanding of this pathway and its interactions with other signaling pathways will advance our ability to develop new interventions towards decreased cancer risk in humans.

Aging↗
PubMed1998

A dominant negative mutant of the insulin-like growth factor-I receptor inhibits the adhesion, invasion, and metastasis of breast cancer.

The 5-year survival rate for women with metastatic breast cancer is only 25-30%; thus, the need to improve treatment is apparent. Overexpression of insulin-like growth factor-I receptor (IGF-IR) correlates with poor prognosis and local recurrence. In this study, we addressed whether functional impairment of IGF-IR affects adhesion, invasion, and metastasis of breast cancer. Impairment of IGF-IR function was achieved by transfecting a dominant negative form of the receptor, termed 486stop, into MDA-MB-435 metastatic breast cancer cells. The protein product of 486stop is secreted extracellularly, resulting in a bystander effect. Cellular adhesion to laminin and collagen was inhibited 94 and 88%, respectively. Furthermore, 486stop inhibited insulin-like growth factor-I-stimulated invasion through collagen IV by 75%. The dominant negative receptor was secreted, as evidenced by the observation that MDA-MB-435 and MDA-MB-231 cells were prevented from binding to laminin by 90% when treated with conditioned medium (CM) from 486stop-transfected cells. CM also inhibited the invasion of MDA-MB-231 cells across collagen IV by 80%. Finally, CM made MDA-MB-231 cells 30% more sensitive to Taxol-induced cell death. Growth in soft agar was suppressed by 486stop, but growth in monolayer was unaffected. When injected into the mammary fat pad, 486stop did not significantly suppress growth of the primary tumor, but metastasis to the lungs, livers, lymph nodes, and lymph vessels was significantly decreased compared to the vector control. In conclusion, inhibition of IGF-IR resulted in suppression of adhesion, invasion, and metastasis, providing a mechanistic rationale for targeting IGF-IR in the treatment of metastatic breast cancer.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Dietary restriction reduces insulin-like growth factor I levels, which modulates apoptosis, cell proliferation, and tumor progression in p53-deficient mice.

Diet contributes to over one-third of cancer deaths in the Western world, yet the factors in the diet that influence cancer are not elucidated. A reduction in caloric intake dramatically slows cancer progression in rodents, and this may be a major contribution to dietary effects on cancer. Insulin-like growth factor I (IGF-I) is lowered during dietary restriction (DR) in both humans and rats. Because IGF-I modulates cell proliferation, apoptosis, and tumorigenesis, the mechanisms behind the protective effects of DR may depend on the reduction of this multifaceted growth factor. To test this hypothesis, IGF-I was restored during DR to ascertain if lowering of IGF-I was central to slowing bladder cancer progression during DR. Heterozygous p53-deficient mice received a bladder carcinogen, p-cresidine, to induce preneoplasia. After confirmation of bladder urothelial preneoplasia, the mice were divided into three groups: (a) ad libitum; (b) 20% DR; and (c) 20% DR plus IGF-I (IGF-I/DR). Serum IGF-I was lowered 24% by DR but was completely restored in the IGF-I/DR-treated mice using recombinant IGF-I administered via osmotic minipumps. Although tumor progression was decreased by DR, restoration of IGF-I serum levels in DR-treated mice increased the stage of the cancers. Furthermore, IGF-I modulated tumor progression independent of changes in body weight. Rates of apoptosis in the preneoplastic lesions were 10 times higher in DR-treated mice compared to those in IGF/DR- and ad libitum-treated mice. Administration of IGF-I to DR-treated mice also stimulated cell proliferation 6-fold in hyperplastic foci. In conclusion, DR lowered IGF-I levels, thereby favoring apoptosis over cell proliferation and ultimately slowing tumor progression. This is the first mechanistic study demonstrating that IGF-I supplementation abrogates the protective effect of DR on neoplastic progression.

Aniline Compounds↗
PubMed1997

Insulin-like growth factor 1 (IGF-1) alters drug sensitivity of HBL100 human breast cancer cells by inhibition of apoptosis induced by diverse anticancer drugs.

In this study, we tested the hypothesis that insulin-like growth factor-1 (IGF-1) modulates apoptosis in human breast cancer cells, HBL100, induced by diverse chemotherapeutic drugs. IGF-1 increased cell survival of HBL100 cells treated with 5-fluorouracil (antimetabolite), methotrexate (antimetabolite), tamoxifen (antiestrogen/antiproliferative), or camptothecin (topoisomerase 1 inhibitor) and after serum withdrawal. Elevated cell survival was not due to an increase in cell proliferation by IGF-1, but rather to an inhibition of apoptosis. Evidence for death by apoptosis was supported by cellular morphology and DNA fragmentation. There were no changes observed in Bcl-2 protein or bax mRNA levels. Extracellular matrix (ECM) is known to influence the apoptotic response of cells; therefore, the antiapoptotic effect of IGF-1 on breast cancer cells was examined using different ECMs: laminin, collagen IV, or Matrigel. IGF-1 protected cells from apoptosis induced by methotrexate on all ECMs tested, providing the first evidence that IGF-1 protects against apoptosis in three-dimensional culture systems. These data provide the rationale to search for drugs that lower serum IGF-1 in an effort to improve the efficacy of chemotherapeutic drugs for the treatment of breast cancer.

Antineoplastic Agents↗
PubMed1997

In vivo antiviral activity and pharmacokinetics of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in woodchuck hepatitis virus-infected woodchucks.

The (-) enantiomer of cis-5-fluoro-1l-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine [(-)-FTC)], a substituted oxathiolane compound with anti-hepatitis B virus activity in vitro, was assessed for its efficacy in woodchucks with naturally acquired woodchuck hepatitis virus (WHV) infection. Pharmacokinetics and in vitro anabolism were also determined. (-)-FTC was anabolized to the 5'-triphosphate in a dose-related fashion, reaching a maximum concentration at about 24 h in cultured woodchuck hepatocytes. Following administration of a dose of 10 mg/kg of body weight intraperitoneally (i.p.), the clearance of (-)-FTC from plasma was monoexponential, the terminal half-life was 3.76 +/- 1.4 h, and the systemic clearance was 0.12 +/- 0.06 liters/h/kg. The antiviral efficacy of (-)-FTC in the woodchuck model was assessed by quantitation of serum WHV DNA levels and by WHV particle-associated DNA polymerase activity at two dosages, 30 and 20 mg/kg given i.p. twice daily (b.i.d.), respectively. The level of WHV DNA in serum was reduced 20- to 150-fold (average, 56-fold) in the 30-mg/kg-b.i.d. treatment group and 6- to 49-fold (average, 27-fold) in the 20-mg/kg-b.i.d. treatment group. Viral DNA polymerase levels diminished accordingly. One week after treatment was discontinued, WHV levels returned to pretreatment levels in both studies. These animals were biopsied before and following treatment with 30 mg of (-)-FTC per kg. Their livers were characterized by a mild increase in cytoplasmic lipid levels, but this change was not associated with altered liver enzyme levels. Serum chemistry and hematology results were within the normal ranges for all treated animals. We conclude that (-)-FTC is a potent antihepadnaviral agent and that it has no detectable toxic effects in woodchucks when given for up to 25 days. Further development of (-)-FTC as an anti-hepatitis B virus therapy for patients is warranted.

Animals↗
PubMed1997

Coordinated expression of myosin heavy chain isoforms and metabolic enzymes within overloaded rat muscle fibers.

We studied the coordinated regulation of myosin heavy chains (MHC) and metabolic enzymes within individual overloaded adult rat plantaris fibers. This was done using monoclonal antibodies raised against distinct developmental and adult MHCs, and quantitative microphotometric succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (GPDH) enzyme assays. Overload shifted MHC expression in the order IIb-->IIx-->IIa-->alpha/I, with a tripling of cells coexpressing I and alpha-MHC, and a transient reexpression of two embryonic MHC and the neonatal isoform in preexisting myofibers. Overload caused a rapid, size-independent, 50% decrease in GPDH activity across all cell types, which recovered by 6 wk. Fiber SDH activities varied according to MHC composition, such that overloaded fibers coexpressing IIa MHC displayed control slow fiber SDH levels, whereas cells expressing IIx and IIb MHC displayed a transient 30% increase in SDH that recovered by 6 wk. Our results suggest that during overload, fibers adapt progressively to the new functional requirements and display more efficient cellular energy utilization and delivery characteristics. The time course of adaptations suggests a role for glycolytic enzymes in the initiation of these transformations.

Adaptation, Physiological↗
PubMed1996

Woodchuck p-glycoprotein found in virus-induced hepatocellular carcinomas binds anticancer drugs.

Virally-induced hepatocellular carcinomas (HCC) are intrinsically resistant to cancer chemotherapy partly due to increased expression of p-glycoprotein (pgp). In this study, we determined that pgp expressed in woodchuck HCC had binding properties were similar to the drug resistant human pgp. Pgp drug binding properties were characterized by photoaffinity labeling with the calcium channel blocker [3H]azidopine (AZD). AZD bound pgp in HCC but not in non-tumor liver samples, and binding was confirmed by competition with Adriamycin (IC50 = 10 microM) and actinomycin D (IC50 = 1 microM). In summary, WHV-induced HCC overexpress a pgp which binds anticancer drugs suggesting a common pathway for drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Regulation of myosin heavy chain expression in adult rat hindlimb muscles during short-term paralysis: comparison of denervation and tetrodotoxin-induced neural inactivation.

The extent to which myosin profiles within adult fast and slow muscles are altered by short-term paralysis remains equivocal. We used an array of specific antibodies to identify adult and developmental MHC isoforms within EDL and soleus muscle fibers, and show a marked multiple expression of MHCs with a general shift towards slower and more energy efficient MHC profiles after 2 weeks of denervation or TTX nerve conduction block. Paralysis also induced marked expression of an embryonic MHC within most EDL cell types, and a subtle, paralysis-sensitive, expression of alpha-cardiac MHC within specific EDL and soleus extrafusal fibers. Comparison of treatment groups also permitted assessment of the relative influence of neural activity versus trophic factors on these isoforms, and confirmed activity as a major, but not sole, regulator of MHC expression.

Animals↗
PubMed1996

Overexpression of a p-glycoprotein in hepatocellular carcinomas from woodchuck hepatitis virus-infected woodchucks (Marmota monax).

The leading cause of human hepatocellular carcinomas (HCCs) is hepatitis B virus (HBV) infection. Woodchucks infected with a closely related hepadnavirus, woodchuck hepatitis virus (WHV), serve as a model for HBV because woodchucks chronically infected with WHV also develop hepatocellular carcinomas. Increased expression of p-glycoprotein (pgp) in human HCCs is a common obstacle in successful cancer chemotherapy. Pgps are encoded by a family of multidrug-resistance (MDR) genes. Livers from uninfected and WHV-infected woodchucks were examined to determine if pgp was expressed in HCCs and if there was a difference in expression between HCCs and nonneoplastic liver. A 170-kd protein was identified by Western blot in HCCs, whereas, constitutive pgp was not detected in normal liver taken from the same animals in 3 of 3 cases. Immunolocalization of the pgp with a panel of monoclonal antibodies revealed intensification of staining in 7 of 20 foci and 12 of 22 HCCs from six animals. Using primers for the human MDR1 gene, a single product was detected by reverse-transcribed polymerase chain reaction (RT-PCR) from HCCs. We have shown an increase in pgp in HCCs compared with normal liver from WHV-infected woodchucks. This is the first example of the induction of a pgp in a naturally hepadnavirus infected rodent system. It suggests the woodchuck can be a useful model for the study of the acquisition of resistance to chemotherapeutic agents in virally induced HCCs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗