Search PubMed⌕ Search

Biomedical subjects

E A Grimm

Publications and source records attributed to E A Grimm.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of iNOS by the p44/42 mitogen-activated protein kinase pathway in human melanoma.

Activating mutations of the genes for NRAS and BRAF, components of the p44/42 mitogen-activated protein kinase (MAPK) pathway, are common findings in melanoma. Recent evidence in several nonmelanoma cell systems supports the regulation of the inducible nitric oxide synthase (iNOS) gene by this pathway. On the basis of our data showing that melanoma iNOS expression predicts shortened patient survival, we formulated the hypothesis that activating mutations of NRAS or BRAF, which lead to constitutive activation of the p44/42 MAPK pathway, drive iNOS expression in human melanoma. In the present study, we have shown that inhibition of melanoma iNOS activity by S-methylisothiourea leads to decreased cell proliferation, confirming the importance of iNOS activity for melanoma cell growth. Regulation of melanoma iNOS expression by the p44/42 MAPK pathway was demonstrated by inhibition of the pathway by U0126, and by BRAF RNA interference. To explore this regulatory pathway in human tissue, 20 melanoma tumors were examined for NRAS and BRAF mutations, immunohistochemical evidence of ERK phosphorylation, and iNOS expression. A significant association was found among these three features. We conclude that in human melanoma, activating mutations of NRAS and BRAF drive constitutive iNOS expression and, implicitly, nitric oxide production, contributing to the poor survival of these patients.

Base Sequence↗
PubMed2001

Down-regulated melanoma differentiation associated gene (mda-7) expression in human melanomas.

The melanoma differentiation associated gene-7 (mda-7) has a potential inhibitory role in melanoma progression, although the mechanisms underlying this effect are still unknown. mda-7 mRNA has been found to be present at higher levels in cultured normal melanocytes compared with metastatic melanoma cell lines. Furthermore, levels of mda-7 message have shown an inverse correlation with melanoma progression in human tumor samples, suggesting that mda-7 may be a novel tumor suppressor gene. We have designed this study to investigate MDA-7 protein expression in different stages of melanoma progression and to examine its antiproliferative effects in vitro. Our data demonstrate that MDA-7 protein can be found in normal melanocytes and early stage melanomas. It is also observed in smooth muscle cells in the skin. However, in keeping with a possible role as a tumor suppressor, MDA-7 expression is decreased in more advanced melanomas, with nearly undetectable levels in metastatic disease. We also investigated antitumor effects of overexpressed MDA-7 on human melanoma cells in vitro. Our results demonstrate that Ad-mda-7 induces apoptosis and G2/M cell cycle arrest in melanoma cells, but not in normal human melanocytes.

Apoptosis↗
PubMed2001

Autoimmunity and hypothyroidism in patients with uveal melanoma.

Among patients with advanced melanoma, the development of autoimmune phenomena or of hypothyroidism during therapy has been associated with a favourable outcome. The objective of this study was to determine the prevalence of autoimmunity and of hypothyroidism in the melanoma population as a whole and to determine if these disease states confer a survival advantage for patients with metastatic disease. We report our findings in the uveal melanoma population. The study population (n = 91) consisted of all patients registered at this institution with the diagnosis of uveal melanoma during a 2 year study period. Eight (8.8%) had a systemic autoimmune disease; 12 (13.2%) were hypothyroid, including 9/46 (19.6%) females. Survival of the stage 4 patients was determined from diagnosis of the primary tumour (SvDx) and from diagnosis of metastatic disease (SvMt), and was compared to that of age/sex matched stage 4 controls. For autoimmune patients versus controls, the median SvDx was 111 months vs 37 months (P = 0.2734) and the median SvMt was 17 months vs 4 months (P = 0.0887). For the hypothyroid patients versus controls, the median SvDx was 58 months vs 49 months (P = 0.5348) and the median SvMt was 4 months vs 8 months (P = 0.2437). We conclude that there is a trend toward longer survival from the date of metastasis in uveal melanoma patients with a systemic autoimmune disorder, suggesting that systemic autoimmunity may play a role in modifying the activity of established metastases. This trend is not seen among the uveal melanoma patients with hypothyroidism. The high prevalence of hypothyroidism suggests a possible molecular interaction between the two disease processes.

Adult↗
PubMed2001

Decreased non-MHC-restricted (CD56+) killer cell cytotoxicity after spaceflight.

Cytotoxic activity of non-major histocompatibility complex-restricted (CD56+) (NMHC) killer cells and cell surface marker expression of peripheral blood mononuclear cells were determined before and after spaceflight. Ten astronauts (9 men, 1 woman) from two space shuttle missions (9- and 10-day duration) participated in the study. Blood samples were collected 10 days before launch, within 3 h after landing, and 3 days after landing. All peripheral blood mononuclear cell preparations were cryopreserved and analyzed simultaneously in a 4-h cytotoxicity (51)Cr release assay using K562 target cells. NMHC killer cell lytic activity was normalized per 1,000 CD56+ cells. When all 10 subjects were considered as one study group, NMHC killer cell numbers did not change significantly during the three sampling periods, but at landing lytic activity had decreased by approximately 40% (P < 0.05) from preflight values. Nine of ten astronauts had decreased lytic activity immediately after flight. NMHC killer cell cytotoxicity of only three astronauts returned toward preflight values by 3 days after landing. Consistent with decreased NMHC killer cell cytotoxicity, urinary cortisol significantly increased after landing compared with preflight levels. Plasma cortisol and ACTH levels at landing were not significantly different from preflight values. No correlation of changes in NMHC killer cell function or hormone levels with factors such as age, gender, mission, or spaceflight experience was found. After landing, expression of the major lymphocyte surface markers (CD3, CD4, CD8, CD14, CD16, CD56), as determined by flow cytometric analysis, did not show any consistent changes from measurements made before flight.

Adrenocorticotropic Hormone↗
PubMed2001

A novel preclinical model of human malignant melanoma utilizing bioreactor rotating-wall vessels.

Malignant melanoma poses a serious health risk which is becoming more crucial as the incidence of this disease steadily increases. The development of appropriate in vitro models that reflect the in vivo tumor environment is a key factor for the study of this malignancy. The local tumor microenvironment plays a critical role in the ability of tumor cells to proliferate and metastasize. While interactions among various cell types are known to be important for tumor growth, most in vitro models utilize only tumor cells, ignoring the importance of tumor-stroma interactions, as well as the contribution of immune cells, which may be important for potential therapies. In addition, the cellular architecture found in vivo, known to be involved in changes in gene expression, is not reflected in standard two-dimensional culture systems. In this study, we have utilized rotating-vessel bioreactors to culture minced human melanoma specimens, allowing the culture of three-dimensional structures which reflect the cellular architecture and heterogeneous composition of the tumor site in vivo. The viability of the pieces in culture can be maintained for 1-2 wk. Immunohistochemical analysis shows multiple cellular types similar to the in vivo situation. Therefore, this system provides a unique model of human melanoma that mimics the in vivo tumor environment much more closely than current culture methods. This novel system may be utilized to determine the mechanism of action of current therapy protocols, as well as to develop new treatment regimens.

Antigens, CD↗
PubMed2000

Characterization of human copine III as a phosphoprotein with associated kinase activity.

The copines, first described by Creutz et al. [(1998) J. Biol. Chem. 273, 1393-1402], comprise a two C2 domain-containing protein family and are known to aggregate phosphatidylserine membranes in a calcium-dependent manner. No enzymatic function has been attributed to copines yet. Due to a cross-reacting activity of Mikbeta1, an antibody to the IL-2Rbeta chain, we were able to serendipitously purify, partially microsequence, and clone human copine III. The 5 kb copine III transcript is expressed ubiquitously as determined by a multitissue Northern blot analysis. Phosphoamino acid analysis revealed phosphorylation of copine III on serine and threonine residues. In vitro kinase assays were performed with immunoprecipitated endogenous copine III, chromatography-purified endogenous copine III, and recombinant copine III expressed in Saccharomyces cerevisiae. The exogenous substrate myelin basic protein was phosphorylated in all in vitro kinase assays containing copine III immunoprecipitate or purified copine III. A 60-kDa band was observed in corresponding in gel kinase assays with staurosporine-activated cells. Cell lines expressing high levels of copine III protein had correspondingly high kinase activity in copine III antiserum immunoprecipitate. However, the copine amino acid sequences lack the traditional kinase catalytic domain. Therefore, the data suggest copine III may possess an intrinsic kinase activity and represent a novel unconventional kinase family.

Amino Acid Sequence↗
PubMed2000

IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway.

IL-2 stimulates extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in various immune cell populations. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK kinase (MKK)/ERK and p38 MAPK pathways are necessary for IL-2 to activate NK cells. Using freshly isolated human NK cells, we established that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK generation, IFN-gamma secretion, and CD25 and CD69 expression. IL-2 induced ERK activation within 5 min. Treatment of NK cells with a specific inhibitor of MKK1/2, PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four sequelae, with inhibition of lymphokine-activated killing induction being least sensitive to MKK/ERK pathway blockade. Activation of p38 MAPK by IL-2 was not detected in NK cells. In contrast to what was observed by others in T lymphocytes, SB203850, a specific inhibitor of p38 MAPK, did not inhibit IL-2-activated NK functions. This data indicate that p38 MAPK activation was not required for IL-2 to activate NK cells for the four functions examined. These results reveal selective signaling differences between NK cells and T lymphocytes; in NK cells, the MKK/ERK pathway and not p38 MAPK plays a critical positive regulatory role during activation by IL-2.

Antigens, CD↗
PubMed2000

Unexpected cytokines in serum of malignant melanoma patients during sequential biochemotherapy.

Biochemotherapy, which combines traditional chemotherapy with immune modulating biologicals, produces an unexpectedly high response rate (>50%) in advanced melanoma patients. We hypothesize that immunological mechanism(s) are responsible for the increased response rate, and particularly that macrophage activation is involved in tumor reduction. Patients were randomized to receive chemotherapy, composed of cisplatin, vinblastine, and dacarbazine (CVD), or biochemotherapy, which is CVD followed by interleukin (IL)-2 and IFN-alpha2b (CVD-BIO). Laboratory analysis was performed on sera from 41 patients from each arm. Measurements of macrophage activation (neopterin), nitric oxide production (nitrite), and tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, IL-1beta, IFN-gamma, IL-6, IL-10, and soluble IL-2 receptor (sIL-2R) were performed. Six of the nine biological responses (nitrite, neopterin, IFN-gamma, IL-6, soluble IL-2R, and IL-10) significantly (P < 0.0002) increased in the CVD-BIO patients but not in the CVD patients. The increased IL-6 (P = 0.04) and IL-10 (P = 0.05) correlated with patient response, but only when the minor responders were included in the analysis. Evidence of macrophage activation was found in CVD-BIO patients and not in those receiving CVD alone. In addition, an unusual cytokine elaboration composed of IL-6, IFN-gamma, IL-10, nitrite, neopterin, and sIL-2R, but not the expected TNF-alpha and IL-1, was detected. A trend of higher increase in IL-6 and IL-10 in patients having clinical response was found, suggesting an incomplete Th2 pattern of cytokine elaboration. These data show that macrophage activation does not appear to be critical in the response to CVD-BIO, but that IL-10 and IL-6 induced by the BIO component of the CVD-BIO were associated with tumor regression, and that their biology should be pursued further in the analysis of mechanism(s) of response.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Inducible nitric oxide synthase and nitrotyrosine in human metastatic melanoma tumors correlate with poor survival.

Despite recognition of the malignant potential of human melanomas, the mechanisms responsible for the pathobiological characteristics contributing to tumor growth, vascular invasiveness, and distant organ metastasis remain undefined. Recent studies have shown that various human tumors express an inducible form of nitric oxide synthase (iNOS) and nitrotyrosine (NT), which suggests a mechanistic role of tumor-associated nitric oxide (NO) in tumorigenesis. We investigated iNOS and NT expression by immunohistochemistry in 20 human metastatic melanoma tissue specimens specifically with respect to iNOS-expressing cell types in the tumor area, pathological and clinical response to systemic therapy, potential role as a prognostic indicator, and NT formation. Our results showed that melanoma cells from 12 of 20 tumors express iNOS, yet the expression of this molecule in the tumor did not correlate with pathological or clinical response to therapy. More importantly, iNOS and NT expression by the melanoma cells strongly correlated with poor survival in patients with stage 3 disease (P < 0.001 and P = 0.020, respectively), suggesting a pathway whereby iNOS might contribute to enhanced tumor progression. In conclusion, our findings strongly suggest that iNOS expression has potential to be considered as a prognostic factor and NO as a critical mediator of an aggressive tumor phenotype in human metastatic melanomas.

Adolescent↗
PubMed1999

Multiple interleukin-2 signaling pathways differentially regulated by microgravity.

Defects in innate immunity have been demonstrated in astronauts after space flight. To investigate the role of microgravity on innate immune function, we evaluated NK and LAK activity of human PBMC stimulated with IL-2 under conditions of simulated microgravity, by using a rotating wall vessel (RWV) culture system. Under these conditions, both NK and LAK activity were generated at levels comparable to those found in static flask cultures. The phenotype of the activated PBMC was similar between the two culture conditions, with one notable exception: the IL-2 receptor alpha chain (CD25), which failed to be upregulated in simulated microgravity. To further investigate this change in IL-2 signaling, we examined the ability of IL-2 to induce secondary cytokines. The production of IFNgamma, IL-1beta, and TNFalpha was almost completely abrogated in the microgravity cultures, suggesting that the IL-2 signaling pathways leading to various IL-2-mediated effects are differentially regulated under bioreactor culture conditions.

Cells, Cultured↗
PubMed1999

Differential increase of Fas ligand expression on metastatic and thin or thick primary melanoma cells compared with interleukin-10.

Fas ligand (FasL), a cell surface molecule belonging to the tumour necrosis factor family, binds to its receptor Fas and thus induces apoptosis of Fas-bearing cells such as activated lymphocytes. In this paper, we report the expression of FasL on melanoma cell lines and patient tumour specimens, and compare it with the expression of interleukin-10 (IL-10), a putative immunosuppressive factor. Apoptosis of Fas-bearing Jurkat cells was increased after interferon-alpha treatment of the FasL-positive melanoma cell line A375, suggesting a regulation of FasL function. We also tested whether FasL and IL-10 were ever co-expressed. Immunohistochemistry studies showed that IL-10 expression was highly positive in the same tumour samples which expressed FasL. In the melanoma patients with thin primaries, 10 of the 12 primaries and six of the seven metastatic lesions were positive for IL-10. In the melanoma patients with thick primaries (> 0.75 mm), four of the five primary lesions and nine of the 10 metastatic lesions were positive for IL-10. In contrast, FasL was generally negative in primary tumours and positive in metastatic tumours. In the thin primary melanoma patients, two of the 12 primaries and five of the seven metastatic tumours were positive for FasL. From the thick melanomas, one of the five primaries and five of the 10 metastatic lesions were positive for FasL. The function of melanoma-derived FasL was confirmed by four different cytotoxicity assays.

Antineoplastic Agents↗
PubMed1998

Variable expression of cytokines in human head and neck squamous cell carcinoma cell lines and consistent expression in surgical specimens.

Head and neck squamous cell carcinomas (HNSCCs) are associated with abnormal cell-mediated immunity at the primary tumor site. We investigated tumor-derived cytokines as factors underlying such abnormalities. Cytokine mRNA and protein of eight HNSCC-derived cell lines were tested; reverse transcription-PCR results indicated the presence of mRNAs for interleukin 1alpha (IL-1alpha) and transforming growth factor alpha (8 of 8); transforming growth factor beta and IL-1beta (7 of 8); and IL-4 and IL-6 (4 of 8). IL-2, IFN-gamma, and tumor necrosis factor alpha mRNA were not detected. Supernatants from six of these cell lines were analyzed by ELISA; IL-1alpha, IL-1beta, and IL-6 were found to be markedly increased compared to human papillomavirus-16-immortalized human oral keratinocytes. To determine whether the cell line findings are applicable to primary tumors, we performed immunohistochemical analysis on tumor specimens from 12 patients with invasive HNSCC. Universal intracellular production of IL-1alpha, IL-1beta, and IL-6 protein was detected. We conclude that the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients.

Adult↗
PubMed1998

Interleukin-1 regulates interleukin-6 secretion in human oral squamous cell carcinoma in vitro: possible influence of p53 but not human papillomavirus E6/E7.

We have previously shown that interleukin-1 (IL-1) and IL-6 are constitutively produced by human oral squamous cell carcinoma (SCC) and some derived cell lines but not by cultured normal oral keratinocytes. To elucidate possible cytokine regulatory pathways that may contribute to oral SCC growth and/or progression, we tested the hypotheses that exogenous and/or endogenous IL-1 regulates IL-6 production in vitro. We investigated the effects of exogenous IL-1 and IL-6 on secondary cytokine secretion. Our studies revealed that IL-1 strongly up-regulated IL-6 protein secretion in all three cell lines tested. This effect was completely abrogated by IL-1 receptor antagonist. IL-1 receptor antagonist also inhibited the secretion of IL-1alpha and IL-1beta in two of three cell lines. These data show for the first time that IL-1 strongly up-regulates IL-6 and support the notion of autocrine regulation of IL-1 in certain oral SCC cell lines. Additionally, because human papillomavirus (HPV) infection and p53 mutation have been implicated in the malignant transformation of SCC, we explored a second hypothesis, that HPV and/or p53 mutation contribute to cytokine dis-regulation. We investigated HPV DNA presence, transcriptional activation of HPV E6/E7 (in HPV DNA-positive cell lines), and p53 gene status in our cell lines. No association between HPV DNA and cytokine expression was found. However, the oral SCC cell lines secreting the most IL-6 had mutant rather than wild-type p53.

Carcinoma, Squamous Cell↗
PubMed1998

Correlation of interleukin 6 with interleukin 1alpha in human mammary tumours, but not with oestrogen receptor expression.

The authors hypothesized that IL-6 production by breast tumour tissues would correlate with OR-positivity, as only those tumours that contain oestrogen receptors (OR) and use oestrogen as a mitogen would benefit from locally increased oestrogen. IL-6 increases the activity of the 17beta-oxidoreductase, which converts oestrone to oestradiol, a process that may contribute to the increased concentration of oestrogen around breast tumours. IL-1alpha upregulates IL-6 production; therefore, the correlation between IL-1alpha and IL-6 immunoreactivity and OR-positivity in paraffin-embedded human breast tumours was further investigated.The results indicate IL-6 immunoreactivity in 40 of 66 paraffin embedded breast tumour specimens, a finding which did not correlate with the clinical evaluation of oestrogen receptor positivity (P=0.32 by Fisher's exact test). However, there was a correlation between IL-6 and IL-1alpha immunoreactivity (P<0.05). To study an in vitro model for this phenomenon, the IL-6 immunoreactivity in available cell lines was tested. Surprisingly, no production of IL-6 protein or mRNA could be detected in any of the cell lines, and this did not change with IL-1alpha stimulation. Therefore, none of the cell lines apparently reflected the immunological potential observed in the majority of surgical specimens.

Breast Neoplasms↗
PubMed1998

Cell cycle regulation of human pancreatic cancer by tamoxifen.

BACKGROUND: Clinical trials have suggested a survival advantage for selected patients with metastatic pancreatic cancer treated with tamoxifen. We sought to identify the molecular mechanism by which tamoxifen inhibits human pancreatic cancer cell (HPCC) growth. METHODS: HPCCs were grown in tamoxifen and growth inhibition was determined by 3H-thymidine uptake and by the MTT assay; changes in cell viability were determined by cell counts. Cell cycle alterations were evaluated by FACS, and the induction of apoptosis was evaluated using the TUNEL assay. Total cellular RNA was isolated after tamoxifen treatment, and Northern blot analysis was performed for p21waf1. RESULTS: Tamoxifen inhibited HPCC growth as measured by inhibition of 3H-thymidine incorporation and by the MTT assay. However, there was no decrease in the total number of viable cells after 6 days of treatment with 10 microM of tamoxifen and no evident apoptosis, confirming the absence of a cytotoxic effect. Cell cycle analysis revealed cellular arrest in the G0/G1 phase, which correlated with p21waf1 mRNA upregulation in response to tamoxifen treatment. CONCLUSIONS: Tamoxifen inhibits HPCC growth by inducing G0/G1 arrest with an associated increase in p21waf1 mRNA expression. Tamoxifen is an effective inhibitor of HPCC growth in vitro and warrants further in vivo study.

Antineoplastic Agents, Hormonal↗
PubMed1998

Suppression of the immune response to an adenovirus vector and enhancement of intratumoral transgene expression by low-dose etoposide.

Adenoviral vectors are commonly used in gene therapy trials because of their efficiency in gene transfer. However, their use is limited by cellular and humoral immune responses that result in temporary transgene expression and reduced efficacy of repeated vector administration. We hypothesized that certain oncolytic agents commonly used to treat cancer patients could suppress the immune response to adenoviral vectors, and enable repeated adenovirus-mediated cancer gene therapy. Etoposide and cyclophosphamide were tested for their ability to suppress the humoral and cellular immune responses to an adenoviral vector in immunocompetent C3H mice. Intratumoral transgene expression was monitored in adenovirus-immunized animals treated with etoposide or cyclophosphamide. Neutralizing antibodies to adenovirus and cytotoxic T lymphocyte (CTL) lysis of virally transduced cells were significantly suppressed in mice treated with etoposide at 2 or 10 mg/kg/day or cyclophosphamide at 10 mg/kg/day compared with untreated mice (P < 0.05). Significantly larger areas of gene transduction were observed in treated animals compared with untreated mice or the mice treated with cyclophosphamide at 2 mg/kg/day (P < 0.05). Our results suggest that repeated adenovirally mediated gene therapy is achievable in cancer patients who are concurrently undergoing treatment with chemotherapy.

Adenoviridae↗
PubMed1998

DNA damage in peripheral blood mononuclear cells correlates with response to biochemotherapy in melanoma.

The combination of cisplatin-based chemotherapy with interleukin-2 (IL-2) and interferon, referred to as biochemotherapy, has shown encouraging results in patients with advanced melanoma. Toxicity is high, however and no objective parameters exist to distinguish between patients who are likely to respond and those who are not. The purpose of this pilot study was to determine whether in vitro cisplatin-induced damage to the glutathione S-transferase-pi (GST-pi) gene in peripheral blood mononuclear cells (PBMCs) before therapy correlated with the histological response in melanoma patients with local-regional metastases who received concurrent biochemotherapy before definitive surgery. Before therapy, PBMCs from 16 patients were exposed to cisplatin at concentrations of 25, 50 or 100 microM for 3 h and the extent of damage to the GST-pi gene was quantitated by polymerase chain reaction (PCR). Patients were subsequently treated on a biochemotherapy regimen consisting of cisplatin 20 mg/m2 intravenously (i.v.) on days 1-4, vinblastine 1.5 mg/m2 i.v. on days 1-4, dacarbazine 800 mg/m2 i.v. on day 1, IL-2 9 MIU/m2 per day i.v. by continuous infusion on days 1-4 (total of 96 h), and interferon alpha2a 5 MU/m2 subcutaneously on days 1-5. The 16 patients were categorized into two groups: major responders (n = 7) and non-major responders (n = 9). Although we observed a wide interpatient variation, a statistically significant correlation existed between the histological response and the degree of DNA damage caused in the PBMCs at all three cisplatin concentrations tested (P = 0.024 for 25 microM; P = 0.036 for 50 microM; P = 0.007 for 100 microM). Our pilot study suggests that determination of in vitro cisplatin-induced DNA damage using a gene-specific PCR assay may be useful in predicting the histological response to biochemotherapy.

Antineoplastic Combined Chemotherapy Protocols↗