Biomedical subjects

R L Baldwin

Publications and source records attributed to R L Baldwin.

At least 19 recordsLinked to original sources

PubMed2000

BRCA1 promoter region hypermethylation in ovarian carcinoma: a population-based study.

There is a clear association between germ-line BRCA1 mutations and inherited ovarian cancer; however, the association between BRCA1 mutations and sporadic ovarian cancer remains ambiguous. The frequency of BRCA1 promoter hypermethylation as an epigenetic means of BRCA1 inactivation was determined for a large, population-based cohort of ovarian cancer patients. BRCA1 promoter hypermethylation was determined by methylation-specific restriction digestion of tumor DNA, followed by Southern blot analysis and confirmed by methylation-specific PCR. BRCA1 promoter hypermethylation was observed in 12 of 98 ovarian tumors. BRCA1 methylation status of the primary tumor was conserved in six recurrent tumors after interim chemotherapy. None of the 12 tumors with BRCA1 promoter hypermethylation demonstrated BRCA1 protein expression by immunohistochemistry. BRCA1 methylation was only seen in ovarian cancer patients without a family history suggestive of a breast/ ovarian cancer syndrome. Therefore, the 12 BRCA1 methylated tumors represented 15% (12 of 81) of the sporadic cancers analyzed in this study. Although the clinical significance of BRCA1 promoter hypermethylation is yet to be determined, promoter hypermethylation may be an alternative to mutation in causing the inactivation of the BRCA1 tumor suppressor gene in sporadic ovarian cancer.

Alleles↗

PubMed2000

Energetics of the interaction between water and the helical peptide group and its role in determining helix propensities.

The alanine helix provides a model system for studying the energetics of interaction between water and the helical peptide group, a possible major factor in the energetics of protein folding. Helix formation is enthalpy-driven (-1.0 kcal/mol per residue). Experimental transfer data (vapor phase to aqueous) for amides give the enthalpy of interaction with water of the amide group as approximately -11.5 kcal/mol. The enthalpy of the helical peptide hydrogen bond, computed for the gas phase by quantum mechanics, is -4.9 kcal/mol. These numbers give an enthalpy deficit for helix formation of -7.6 kcal/mol. To study this problem, we calculate the electrostatic solvation free energy (ESF) of the peptide groups in the helical and beta-strand conformations, by using the delphi program and parse parameter set. Experimental data show that the ESF values of amides are almost entirely enthalpic. Two key results are: in the beta-strand conformation, the ESF value of an interior alanine peptide group is -7.9 kcal/mol, substantially less than that of N-methylacetamide (-12.2 kcal/mol), and the helical peptide group is solvated with an ESF of -2.5 kcal/mol. These results reduce the enthalpy deficit to -1.5 kcal/mol, and desolvation of peptide groups through partial burial in the random coil may account for the remainder. Mutant peptides in the helical conformation show ESF differences among nonpolar amino acids that are comparable to observed helix propensity differences, but the ESF differences in the random coil conformation still must be subtracted.

Alanine↗

PubMed2000

The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry.

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.

Apoproteins↗

PubMed2000

Effects of diet forage:concentrate ratio and metabolizable energy intake on visceral organ growth and in vitro oxidative capacity of gut tissues in sheep.

We used 28 crossbred wether lambs to determine the effects of dietary forage:concentrate ratio and metabolizable energy intake on visceral organ growth and oxidative capacity of gut tissues in lambs. Lambs were assigned randomly to a factorial arrangement of dietary treatments consisting of pelleted diets containing either 75% orchardgrass or 75% concentrate fed once daily at either .099 or .181 Mcal ME x (kg BW(.75))(-1) x d(-1). After a 52-d feeding period, lambs were slaughtered to obtain measurements of visceral organ mass and composition and oxidative capacity of isolated epithelial cells. Lamb performance, as measured by DMI, ADG, and efficiency of gain, was greater (P = .0001) for both diets at high ME intake. Likewise, lambs fed 75% concentrate gained faster and more (P < or = .01) efficiently than lambs fed 75% forage. Total digestive tract (TDT; includes rumen, reticulum, omasum, abomasum, and intestines) weight increased (P = .0001) with ME intake and was greater (P = .03) in lambs fed 75% forage than in those fed 75% concentrate. As a percentage of empty body weight (EBW), TDT weight increased with ME intake in lambs fed 75% forage, but it was unaffected by ME intake in lambs fed 75% concentrate (diet x intake, P = .03). Liver weight increased (P = .0001) with ME intake and was greater (P = .005) in lambs fed 75% concentrate vs 75% forage; however, liver weight as a percentage of EBW was increased (P = .0002) with ME intake but was unaffected by diet. Greater ME intake increased (P < or = .02) small intestinal (SI) epithelial and muscle mass of 15-cm sections, whereas jejunal epithelial mass was greater (P = .01) for lambs fed 75% forage vs 75% concentrate. Rumen epithelial concentrations of DNA and RNA increased (P < or = .02) with greater ME intake, whereas SI concentrations of DNA and RNA were largely unaffected by diet or ME intake. The activity of Na(+)-K(+)-ATPase increased in ileal epithelium (P < or = .02) with ME intake and concentrate in the diet, but activity in ruminal epithelium increased (P = .05) with concentrate. Total oxygen consumption by isolated ruminal and intestinal epithelial cells was unaffected by treatment. These data suggest that ME intake and level of dietary forage affect ruminal and intestinal growth via changes in cellular hyperplasia. Additionally, this study supports the concept that ME intake and diet composition alter gut energy expenditure, at least in part, through changes in mass rather than mass specific metabolism.

Animal Feed↗

PubMed2000

Effects of diet forage:concentrate ratio and metabolizable energy intake on isolated rumen epithelial cell metabolism in vitro.

Crossbred wether lambs were used to assess the effect of altered forage:concentrate ratio and metabolizable energy intake on metabolism of substrates by ruminal epithelium using an isolated cell system. Lambs (n = 28; 20.1 +/- 3 kg BW) were assigned randomly to a factorial arrangement of dietary treatments consisting of either 75% forage or 75% concentrate fed once daily at either .099 or .181 Mcal ME x(kg BW(.75))(-1) x d(-1) for 52 d. After a 52-d feeding period, isolated rumen epithelial cells (IREC) were incubated in the presence of an oxidizable substrate with a single 14C label (acetate, propionate, butyrate, glucose, glutamate, and glutamine) at concentrations ranging from .1 to 50 mM, and substrate oxidation to 14CO2 or metabolism to beta-hydroxybutyrate (beta-HBA), acetoacetate, pyruvate, and lactate was determined. For all substrates, oxidation to CO2 was concentration-dependent and saturable within the physiological range. Differences in substrate oxidation to CO2 by IREC at specific substrate concentrations did not affect Vmax (maximal rate of substrate oxidation, nmol oxidized to CO2 x 1 x 10(6) cells(-1) x 90 min(-1)) and K(ox) (concentration of substrate at which half Vmax oxidation rate is achieved, mmoles/L) estimates for the dietary treatments. Production of beta-HBA from butyrate by IREC from the lambs fed 75% forage was not affected by ME intake; however, production was elevated by high ME intake of the 75% concentrate diet (diet x intake interaction; P < .02). Acetoacetate production from butyrate by IREC from lambs fed at high ME intake was greater (P = .001) than from those fed at low ME intake. Lactate and pyruvate production from glucose, glutamate, and propionate were generally unaffected by dietary treatment; however, rate of glutamine metabolism to lactate and pyruvate by IREC was increased with increased ME intake. The observed changes in metabolite production rates across groups did not affect the predicted Vmax and K(ox) parameter estimates. The estimated K(ox) values corroborate that VFA are the primary oxidizable fuels used by ruminal epithelial cells while illustrating that other substrates such as glucose, glutamate, and glutamine would not be expected to be oxidized extensively in vivo due to the high K(ox) relative to substrate concentrations in vivo. In conclusion, the capacity of isolated ruminal epithelial cells to oxidize substrates was largely unaffected by ME intake or dietary forage:concentrate ratio of the diet.

Animal Feed↗

PubMed2000

Sheep rumen metabolic development in response to age and dietary treatments.

This study examined the time course of rumen metabolic development in the absence of solid feed consumption and the effect of delayed solid feed consumption on sheep rumen development. Twenty-seven lambs consumed milk replacer until slaughter at nine ages from 1 to 84 d (milk group). Three additional lambs consumed milk replacer from 1 to 48 d. From 49 d until slaughter at 84 d, these lambs were weaned onto solid feed (fed group). At slaughter, rumen contents were removed for VFA analysis and rumen epithelium was preserved for morphological examination. Rumen epithelial cells were isolated and incubated in media containing 2.5 mM U-[14C]-glucose or 10 mM 1-[14C]-butyrate. Rumen VFA concentrations did not change with age in lambs given milk replacer. At 84 d of age, intraruminal VFA concentrations were elevated in lambs consuming solid feed compared to 84-d-old lambs given milk replacer (P < .05). The number of ruminal papillae per square centimeter decreased (P < .05) while papillae length and width did not change significantly with age in rumen epithelium from lambs given milk replacer. At 84 d of age, rumen epithelium from lambs in the fed group had fewer and larger papillae/per square centimeter than rumen epithelium from lambs given milk replacer (P < .05). Rates of glucose and butyrate oxidation and acetoacetate and lactate production by rumen cells isolated from lambs given milk replacer did not change with age. Beta-hydroxybutyrate (BHBA) production was undetectable before 42 d of age in lambs given milk replacer and increased to levels found in conventionally raised adults by 84 d. At 84 d there were no differences in rates of glucose and butyrate oxidation or acetoacetate and lactate production by rumen cells between the two treatment groups. Thus, the change in substrate oxidation from glucose to butyrate, indicative of rumen metabolic maturation, does not occur in the absence of solid feed consumption. However, the development of rumen ketogenesis, as evidenced by increased BHBA production, does occur in the absence of solid feed consumption. Delaying the initiation of solid feed consumption results in rumen morphological development but does not stimulate rumen metabolic development. Increased intraruminal VFA concentrations, earlier exposure to VFA, or a longer period of exposure to VFA may be required to induce the genes responsible for rumen metabolic development.

Aging↗

PubMed2000

Estimation of parameters describing lipid metabolism in lactation: challenge of existing knowledge described in a model of metabolism.

We have been conducting research to improve quantitative descriptions of metabolism in lactating dairy cows depicted in an existing mechanistic, computer-assisted model. The model is dynamic and deterministic and is based on biochemical equations describing ruminal fermentation and chemical interactions in body tissues. The objective was to challenge this model with data collected in in vivo and in vitro experiments on high producing dairy cattle fed a range of energy. Cows that varied in genetic propensity for milk production (7045 to 12,909 kg of milk/305 d), lactation number (1 to 4), stage of lactation (-30 to 345 d in milk), and rate of intake (14 to 29 kg/d of dry matter) and that were fed various energy-yielding feedstuffs were used. Dietary inputs, milk component outputs, body fat, nutrient concentrations in blood, and maximal velocity and substrate sensitivity of adipose tissue metabolic reactions were observed. Model simulations were conducted; simulated yields of milk components for a 305-d lactation were within 5% of observed means. Simulated lipid metabolism and accumulation of body fat were adequate in many situations; however, the model response to changes in energy intake was too sensitive. This inadequacy was especially noticeable in later lactation because of inadequate representation of dynamic responses over periods more than a few weeks long. The model behaves consistently with biochemical principles, behavior was in the correct direction, and precision was adequate for many variables. Lack of precision in long-term dynamic changes indicates that the parameters describing energy-utilizing reactions are inadequate. This severe challenge of the model supports its functionality. Further experiments must be designed to determine how nutrients in viscera, muscle, and adipose tissue are used; these experiments must encompass sufficient range in genetic ability, nutrient input, and time to adequately describe the dynamic and integrated nature of metabolic reactions.

Adipose Tissue↗

PubMed1999

Comparative hybridization of an array of 21,500 ovarian cDNAs for the discovery of genes overexpressed in ovarian carcinomas.

Comparative hybridization of cDNA arrays is a powerful tool for the measurement of differences in gene expression between two or more tissues. We optimized this technique and employed it to discover genes with potential for the diagnosis of ovarian cancer. This cancer is rarely identified in time for a good prognosis after diagnosis. An array of 21,500 unknown ovarian cDNAs was hybridized with labeled first-strand cDNA from 10 ovarian tumors and six normal tissues. One hundred and thirty-four clones are overexpressed in at least five of the 10 tumors. These cDNAs were sequenced and compared to public sequence databases. One of these, the gene HE4, was found to be expressed primarily in some ovarian cancers, and is thus a potential marker of ovarian carcinoma.

Biomarkers, Tumor↗

PubMed1999

The 28-111 disulfide bond constrains the alpha-lactalbumin molten globule and weakens its cooperativity of folding.

Our aim is to determine whether the disulfide bonds of alpha-lactalbumin account for the lack of cooperative folding behavior reported for some molten globule variants, in contrast to the highly cooperative folding reported for the pH 4 molten globule of apomyoglobin. Two different alpha-lactalbumin genetic constructs are studied: [28-111], which has a single disulfide bond connecting two segments of the alpha-helix domain, and [all-Ala], which has no disulfide bonds. The superposition test used earlier to probe for cooperative folding of the apomyoglobin molten globule is used to determine whether there is an important difference in folding cooperativity between the molten globules of [28-111] and [all-Ala]. The [all-Ala] construct behaves in the same manner as the apomyoglobin molten globule: its folding satisfies the superposition test in the three sets of anion conditions studied, and anions stabilize it against urea unfolding. The [28-111] construct behaves differently in both respects: the folding of its molten globule does not satisfy the superposition test in two of the three sets of anion conditions, and anions barely affect its stability. The 28-111 disulfide bond stabilizes the molten globule substantially, as expected from earlier work. Comparison of the unfolding transition curves monitored by circular dichroism also demonstrates that [28-111] folds in a less cooperative manner than [all-Ala]: the unfolding curve of [28-111] is significantly broader. Moreover, the unfolding curves indicate that [28-111] has a lower helix content than [all-Ala]. Consequently, the 28-111 bond constrains the folding behavior of the molten globule and weakens its cooperativity of folding.

Disulfides↗

PubMed1999

Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate.

Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia equilibrium Ib. In urea-induced unfolding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib-->Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U equilibrium Ia equilibrium Ib equilibrium N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2. 7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.

Animals↗

PubMed1999

Putative interhelix ion pairs involved in the stability of myoglobin.

An earlier theoretical study predicted that specific ion pair interactions between neighboring helices should be important in stabilizing myoglobin. To measure these interactions in sperm whale myoglobin, single mutations were made to disrupt them. To obtain reliable DeltaG values, conditions were found in which the urea induced unfolding of holomyoglobin is reversible and two-state. The cyanomet form of myoglobin satisfies this condition at pH 5, 25 degrees C. The unfolding curves monitored by far-UV CD and Soret absorbance are superimposable and reversible. None of the putative ion pairs studied here makes a large contribution to the stability of native myoglobin. The protein stability does decrease somewhat between 0 and 0.1 M NaCl, however, indicating that electrostatic interactions contribute favorably to myoglobin stability at pH 5.0. A previous mutational study indicated that the net positive charge of the A[B]GH subdomain of myoglobin is an important factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain do not contribute significantly to its stability. One of the assumptions made in that study is tested here: replacement of either positively or negatively charged residues outside the A[B]GH subdomain has no significant effect on the stability of the pH 4 molten globule.

Animals↗

PubMed1999

Interaction between water and polar groups of the helix backbone: an important determinant of helix propensities.

We report an enthalpic factor involved in determining helix propensities of nonpolar amino acids. Thermal unfolding curves of the five 13-residue peptides, Ac-KA4XA4KGY-NH2 (X = Ala, Leu, Ile, Val, Gly), have been measured by using CD in water/trifluoroethanol (TFE) mixtures. The peptide helix contents show that the rank order of helix propensities changes with temperature: although Ala has the highest helix propensity at 0 degrees C in all TFE concentrations, it is lower than Leu, Ile, and Val at 50 degrees C in 20% TFE. This change is attributed to shielding by nonpolar side chains of the interaction between water and polar groups in the helix backbone for the following reasons. (i) Helix content is directly related to helix propensity for these designed peptides because side-chain-side-chain interactions are absent. (ii) The change in rank order with temperature is enthalpic in origin: in water, the apparent enthalpy of helix formation calculated from the thermal unfolding curves varies widely among the five peptides and has the same rank order as the helix propensities at 0 degrees C. The rank order does not result from burial of nonpolar surface area because the calculated heat capacity change (DeltaCp) on helix formation is opposite in sign from the expected DeltaCp. (iii) A nonpolar side chain can exclude water from interacting with helix polar groups, according to calculations of water-accessible surface area, and the polar interaction between water and peptide polar groups is entirely enthalpic, as shown by amide transfer data.

Amino Acids↗

PubMed1999

Alanine is helix-stabilizing in both template-nucleated and standard peptide helices.

Alanine-based peptides of defined sequence and length show measurable helix contents, allowing them to be used as a model system both for analyzing the mechanism of helix formation and for investigating the contributions of side-chain interactions to protein stability. Extensive characterization of many peptide sequences with varying amino acid contents indicates that the favorable helicity of alanine-based peptides can be attributed to the large helix-stabilizing propensity of alanine. Based on their analysis of alanine-rich sequences N-terminally linked to a synthetic helix-inducing template, Kemp and coworkers [Kemp, D. S., Boyd, J. G. & Muendel, C. C. (1991) Nature (London) 352, 451-454; Kemp, D. S., Oslick, S. L. & Allen, T. J. (1996) J. Am. Chem. Soc. 118, 4249-4255] argue that alanine is helix-indifferent, however, and that the favorable helix contents of alanine-based peptides must have some other explanation. Here, we show that the helix contents of template-nucleated sequences are influenced strongly by properties of the template-helix junction. A model in which the helix propensities of residues at the template-peptide junction are treated separately brings the results from alanine-based peptides and template-nucleated helices into agreement. The resulting model provides a physically plausible resolution of the discrepancies between the two systems and allows the helix contents of both template-nucleated and standard peptide helices to be predicted by using a single set of helix propensities. Helix formation in both standard peptides and template-peptide conjugates can be attributed to the large intrinsic helix-forming tendency of alanine.

Alanine↗

PubMed1999

Specificity of native-like interhelical hydrophobic contacts in the apomyoglobin intermediate.

On exposure to mildly acidic conditions, apomyoglobin forms a partially folded intermediate, I. The A, B, G, and H helices are significantly structured in this equilibrium intermediate, whereas the remainder of the protein is largely unfolded. We report here the effects of mutations at helix pairing sites on the stability of I in three classes of mutants that: (i) truncate hydrophobic side chains in native helix packing sites, (ii) truncate hydrophobic side chains not involved in interhelical contacts, and (iii) extend hydrophobic side chains at residues not involved in interhelical contacts. Class I mutants significantly decrease the stability and cooperativity of folding of the intermediate. Class II and III mutants show smaller effects on stability and have little effect on cooperativity. Qualitatively similar results to those found in I were obtained for all three classes of mutants in native myoglobin (N), demonstrating that hydrophobic burial is fairly specific to native helix packing sites in I as well as in N. These results suggest that hydrophobic burial along native-like interhelical contacts is important for the formation of the cooperatively folded intermediate.

Amino Acid Sequence↗

PubMed1999

A specific transition state for S-peptide combining with folded S-protein and then refolding.

We measured the folding and unfolding kinetics of mutants for a simple protein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form ribonuclease S analogues: initially, S-peptide is disordered whereas S-protein is folded. The fluorescent probe provides a convenient spectroscopic probe for the reaction. The association rate constant, kon, and the dissociation rate constant, koff, were both determined for two sets of mutants. The dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows kon and koff to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding and refolding. The first set of mutants tests the role of the alpha-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the alpha-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log kon vs. log Kd for this series of mutants is linear over a very wide range, with a slope of -0.3, indicating that almost all of the molecules fold via a transition state involving the helix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, which are known to be important for binding S-peptide to S-protein and which also contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transition state. The results provide a remarkably clear description of a folding transition state.

Amino Acid Sequence↗

PubMed1999

Altered expression of transforming growth factor-beta ligands and receptors in primary and recurrent ovarian carcinoma.

BACKGROUND: Resistance to the potent growth inhibitory effects of transforming growth factor-beta (TGF-beta) is a characteristic of many malignancies. TGF-beta insensitivity has been attributed to alterations in the number and function of the TGF-beta receptors as well as disturbances of downstream signal transduction. Paradoxically, increased levels of TGF-beta ligand have been demonstrated in several types of malignant tumors. TGF-beta also may play a role in ovarian carcinogenesis; however, the nature of this interaction has yet to be defined completely. METHODS: To explore the potential role of TGF-beta-mediated autocrine and paracrine influences in epithelial ovarian carcinoma, mRNA expression levels of the three TGF-beta ligand isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) and the three TGF-beta receptors (TbetaR-I, T/betaR-II, and TbetaR-III) were examined by Northern blot analysis in both primary and recurrent ovarian carcinoma specimens. Immunohistochemical analysis was performed to localize expression of TbetaR-I and TbetaR-II, whereas the presence of genetic alterations in TbetaR-1 was examined through Southern blot analysis. RESULTS: Compared with normal ovarian tissue, both primary and recurrent ovarian carcinomas demonstrated significant overexpression of the TGF-beta1 and TGF-beta3 mRNA transcripts. TGF-beta2 expression was detectable in 75% of primary and only 53% of recurrent tumor specimens. Alterations also were detected in TbetaR mRNA expression. Expression levels of TbetaR-III were significantly reduced in both primary and recurrent ovarian carcinomas. Furthermore, detectable levels of TbetaR-I and TbetaR-III mRNA transcripts were present in only 47% and 50% of recurrent ovarian tumors, respectively. Immunohistochemical staining demonstrated that TbetaR-I and TbetaR-II expression localized to tumor cells; however, receptor staining in stromal tissue also was detected. Southern blot analysis of TbetaR-I did not reveal any major genetic changes to account for the absence of TbetaR-I expression. CONCLUSIONS: Alterations in expression of TGF-beta ligands and receptors consistently were greater in recurrent ovarian carcinomas compared with primary tumors, and may reflect a phenotype that promotes tumor recurrence or chemoresistance. Together, these data suggest that enhanced expression of TGF-betaI and TGF-beta3, as well as the loss of expression of TbetaR-I and TbetaR-III, contribute to ovarian carcinogenesis and/or tumor progression.

Blotting, Northern↗

PubMed1999

Primary ovarian cancer cultures are resistant to Fas-mediated apoptosis.

OBJECTIVES: Fas, a primary mediator of cellular apoptosis, is expressed by the normal ovarian epithelium. We analyzed the levels of Fas and soluble Fas (sFas) expression in ovarian cancer tissue and determined the susceptibility of primary ovarian cancer cell (CSOC) cultures to Fas-mediated apoptosis. METHODS: Fas mRNA levels were detected by RT-PCR, and Fas protein levels were determined by immunohistochemistry and Western blot analysis. Secreted sFas levels were measured by ELISA. Localization of Fas to the cell surface was demonstrated by flow cytometry. The effect of Fas on cell proliferation was measured by MTT assay. RESULTS: Intense Fas staining was detected on the cell surface and in the cytoplasm of ovarian carcinoma specimens. We also found that mean levels of sFas, which can function as a Fas agonist, were significantly increased in 18 sera from cancer patients (0.98 ng/ml) compared to those of 8 healthy individuals (0.61 ng/ml, P = 0.004). Fas mRNA and protein were expressed in all primary ovarian cancer cell cultures. Despite abundant Fas expression, CSOC cultures were significantly less sensitive to Fas-mediated apoptosis (11.3%) than primary cultures of normal ovarian epithelial cells (HOSE) (50.0%) (P = 0.00001). The sFas level in CSOC-conditioned medium was minimal (0.07 ng/ml) and not significantly different from that of HOSE-conditioned medium (0. 09 ng/ml). The small amount of sFas secreted by CSOC does not likely account for the observed resistance to Fas-mediated apoptosis. CONCLUSION: Decreased sensitivity to Fas-mediated apoptosis could contribute to ovarian tumorigenesis through resistance to cytotoxic T lymphocyte-mediated cytotoxicity and may play a role in ovarian tumorigenesis.

Apoptosis↗

PubMed1999

Ovarian carcinoma cell cultures are resistant to TGF-beta1-mediated growth inhibition despite expression of functional receptors.

OBJECTIVE: The purpose of this study was to determine the response of ovarian carcinoma cells to TGF-beta1 and to examine components of the TGF-beta signaling pathway. METHODS: Twenty-three primary ovarian cancer cell (CSOC) cultures established from solid ovarian carcinomas were treated with TGF-beta1 and assayed for growth response by MTT assay. Expression of TGF-beta receptor I (TbetaR-I) and receptor II (TbetaR-II), essential for effective signaling, was determined by Western analysis of CSOC cultures. TGF-beta1 ligand-induced phosphorylation of TbetaR-I was determined by immunoprecipitation of TbetaR-I followed by a protein kinase assay to assess TbetaR-I phosphorylation, an essential first step in TGF-beta signal transduction. Gelatin zymography performed on 5 CSOC cultures incubated with TGF-beta1 was used to determine TGF-beta's effect on matrix metalloproteinase production. Normal ovarian surface epithelial cells were used for comparison. RESULTS: Eighteen of twenty-three (78%) CSOC cultures demonstrated no significant growth inhibition in response to TGF-beta1 treatment. All cell cultures expressed TbetaR-I and TbetaR-II and exhibited TbetaR-I phosphorylation following TGF-beta1 treatment. CSOC cultures produced significantly higher levels of matrix metalloproteinase-2 (MMP-2) than normal ovarian surface epithelial cells; however, the level of MMP-2 expression was not regulated by TGF-beta1. CONCLUSION: These results indicate that TGF-beta1 resistance and higher levels of MMP-2 production may be inherent properties of the ovarian cancer phenotype. The initial steps in the TGF-beta signaling pathway, receptor expression, ligand binding, and TbetaR-I phosphorylation, appear to be functional in primary ovarian cancer cell cultures. Therefore, the mechanism of growth resistance is downstream of TbetaR-I phosphorylation.

Cell Division↗