Infections by Renibacterium salmoninarum and Nanophyetus salmincola Chapin are associated with reduced growth of juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), in the Northeast Pacific Ocean.

We examined 1454 juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), captured in nearshore waters off the coasts of Washington and Oregon (USA) from 1999 to 2004 for infection by Renibacterium salmoninarum, Nanophyetus salmincola Chapin and skin metacercariae. The prevalence and intensities for each of these infections were established for both yearling and subyearling Chinook salmon. Two metrics of salmon growth, weight residuals and plasma levels of insulin-like growth factor-1, were determined for salmon infected with these pathogens/parasites, both individually and in combination, with uninfected fish used for comparison. Yearling Chinook salmon infected with R. salmoninarum had significantly reduced weight residuals. Chinook salmon infected with skin metacercariae alone did not have significantly reduced growth metrics. Dual infections were not associated with significantly more severe effects on the growth metrics than single infections; the number of triple infections was very low and precluded statistical comparison. Overall, these data suggest that infections by these organisms can be associated with reduced juvenile Chinook salmon growth. Because growth in the first year at sea has been linked to survival for some stocks of Chinook salmon, the infections may therefore play a role in regulating these populations in the Northeast Pacific Ocean.

Differential growth in estuarine and freshwater habitats indicated by plasma IGF1 concentrations and otolith chemistry in Dolly Varden Salvelinus malma.

This study employed a combination of otolith microchemistry to indicate the recent habitat use, and plasma concentrations of the hormone insulin-like growth factor 1 (IGF1) as an index of recent growth rate, to demonstrate differences in growth and habitat use by Dolly Varden Salvelinus malma occupying both freshwater and estuarine habitats in south-west Alaska. Extensive sampling in all habitats revealed that fish had higher IGF1 levels in estuarine compared to lake habitats throughout the summer, and that the growth rates in different habitats within the estuary varied seasonally. In addition, otolith microchemistry indicated differentiation in estuarine habitat use among individual S. malma throughout summer months. Although growth in the estuary was higher than in fresh water in nearly all sites and months, the benefits and use of the estuarine habitats varied on finer spatial scales. Therefore, this study further illustrates the diverse life histories of S. malma and indicates an evaluation of the benefits of marine waters needs to include sub-estuary scale habitat use.

Growth and endocrine effects of recombinant bovine growth hormone treatment in non-transgenic and growth hormone transgenic coho salmon.

To examine the relative growth, endocrine, and gene expression effects of growth hormone (GH) transgenesis vs. GH protein treatment, wild-type non-transgenic and GH transgenic coho salmon were treated with a sustained-release formulation of recombinant bovine GH (bGH; Posilac). Fish size, specific growth rate (SGR), and condition factor (CF) were monitored for 14 weeks, after which endocrine parameters were measured. Transgenic fish had much higher growth, SGR and CF than non-transgenic fish, and bGH injection significantly increased weight and SGR in non-transgenic but not transgenic fish. Plasma salmon GH concentrations decreased with bGH treatment in non-transgenic but not in transgenic fish where levels were similar to controls. Higher GH mRNA levels were detected in transgenic muscle and liver but no differences were observed in GH receptor (GHR) mRNA levels. In non-transgenic pituitary, GH and GHR mRNA levels per mg pituitary decreased with bGH dose to levels seen in transgenic salmon. Plasma IGF-I was elevated with bGH dose only in non-transgenic fish, while transgenic fish maintained an elevated level of IGF-I with or without bGH treatment. A similar trend was seen for liver IGF-I mRNA levels. Thus, bGH treatment increased fish growth and influenced feedback on endocrine parameters in non-transgenic but not in transgenic fish. A lack of further growth stimulation of GH transgenic fish suggests that these fish are experiencing maximal growth stimulation via GH pathways.

Comparative evaluation of vaccine efficacy of recombinant Marek's disease virus vaccine lacking Meq oncogene in commercial chickens.

Marek's disease virus (MDV) oncogene meq has been identified as the gene involved in tumorigenesis in chickens. We have recently developed a Meq-null virus, rMd5 Delta Meq, in which the oncogene meq was deleted. Vaccine efficacy experiments conducted in Avian Disease and Oncology Laboratory (ADOL) 15I(5) x 7(1) chickens vaccinated with rMd5 Delta Meq virus or an ADOL preparation of CVI988/Rispens indicated that rMd5 Delta Meq provided superior protection than CVI988/Rispens when challenged with the very virulent plus MDV 648A strain. In the present study we set to investigate the vaccine efficacy of rMd5 Delta Meq in the field compared to several commercial preparations of CVI988/Rispens. Three large-scale field experiments, in which seeder chickens were inoculated with a very virulent plus strain of 686, vv+ MDV, were conducted in a model developed by Hy-Line International. In addition, comparisons were made with bivalent vaccine (HVT+SB-1), HVT alone and several serotype 3 HVT-vectored vaccines individually or in combination with CVI988/Rispens. Experimental results showed that addition of HVT to either of the two commercial CVI988/Rispens preparations tested (A or B) did not enhance protection conferred by CVI988/Rispens alone and that rMd5 Delta Meq was a better or equal vaccine compared to any of the CVI988/Rispens vaccines tested under the conditions of the field trials presented herein. Our results also emphasized the complexity of factors affecting vaccine efficacy and the importance of challenge dose in protection.

Footwear and running cardio-respiratory responses.

This study compared cardio-respiratory responses during running wearing a motion control shoe (MC) or a cushioning shoe (CU) in a cross-over single blinded design. Fourteen runners (10F/4M, age=27.3+/-5.1 years, body mass=64.1+/-12.2 kg, height=167.8+/-7.5 cm, VO (2)max=52.3+/-8.8 ml/kg/min) completed a 40-min run at approximately 65% VO (2) max under both shoe conditions. Oxygen uptake (mL/kg/min; L/min), minute ventilation (L/min), respiratory exchange ratio, and heart rate were measured at minutes 8-10, 18-20, 28-30 and 38-40 of exercise. Rating of perceived exertion was obtained at minutes 10, 20, 30 and 40. Two (footwear) by four (time) repeated measures ANOVAs showed no differences between footwear conditions in overall oxygen consumption (MC=36.8+/-1.5 vs. CU=35.3+/-1.4 mL/kg/min, p=0.143), minute ventilation (MC=50.4+/-4 vs. CU=48.5+/-3.8, p=0.147), respiratory exchange ratio (MC=0.90+/-0.01 vs. CU=0.89+/-0.01, p=0.331), heart rate (MC=159+/-3 vs. CU=160+/-3, p=0.926), or rate of perceived exertion. The design of motion control footwear does not appear to affect cardio-respiratory or perceived exertion responses during submaximal running. The findings are specific to the shoes tested. Nonetheless, the outcomes suggest that footwear selection to reduce certain overuse injuries does not increase the work of running.

Endocrine effects of growth hormone overexpression in transgenic coho salmon.

Non-transgenic (wild-type) coho salmon (Oncorhynchus kisutch), growth hormone (GH) transgenic salmon (with highly elevated growth rates), and GH transgenic salmon pair fed a non-transgenic ration level (and thus growing at the non-transgenic rate) were examined for plasma hormone concentrations, and liver, muscle, hypothalamus, telencephalon, and pituitary mRNA levels. GH transgenic salmon exhibited increased plasma GH levels, and enhanced liver, muscle and hypothalamic GH mRNA levels. Insulin-like growth factor-I (IGF-I) in plasma, and growth hormone receptor (GHR) and IGF-I mRNA levels in liver and muscle, were higher in fully fed transgenic than non-transgenic fish. GHR mRNA levels in transgenic fish were unaffected by ration-restriction, whereas plasma GH was increased and plasma IGF-I and liver IGF-I mRNA were decreased to wild-type levels. These data reveal that strong nutritional modulation of IGF-I production remains even in the presence of constitutive ectopic GH expression in these transgenic fish. Liver GHR membrane protein levels were not different from controls, whereas, in muscle, GHR levels were elevated approximately 5-fold in transgenic fish. Paracrine stimulation of IGF-I by ectopic GH production in non-pituitary tissues is suggested by increased basal cartilage sulphation observed in the transgenic salmon. Levels of mRNA for growth hormone-releasing hormone (GHRH) and cholecystokinin (CCK) did not differ between groups. Despite its role in appetite stimulation, neuropeptide Y (NPY) mRNA was not found to be elevated in transgenic groups.

Previtellogenic oocyte growth in salmon: relationships among body growth, plasma insulin-like growth factor-1, estradiol-17beta, follicle-stimulating hormone and expression of ovarian genes for insulin-like growth factors, steroidogenic-acute regulatory protein and receptors for gonadotropins, growth hormone, and somatolactin.

Body growth during critical periods is known to be an important factor in determining the age of maturity and fecundity in fish. However, the endocrine mechanisms controlling oogenesis in fish and the effects of growth on this process are poorly understood. In this study interactions between the growth and reproductive systems were examined by monitoring changes in various components of the FSH-ovary axis, plasma insulin-like growth factor 1 (Igf1), and ovarian gene expression in relation to body and previtellogenic oocyte growth in coho salmon. Samples were collected from females during two hypothesized critical periods when growth influences maturation in this species. Body growth during the fall-spring months was strongly related to the degree of oocyte development, with larger fish possessing more advanced oocytes than smaller, slower growing fish. The accumulation of cortical alveoli in the oocytes was associated with increases in plasma and pituitary FSH, plasma estradiol-17beta, and ovarian steroidogenic acute regulatory protein (star) gene expression, whereas ovarian transcripts for growth hormone receptor and somatolactin receptor decreased. As oocytes accumulated lipid droplets, a general increase occurred in plasma Igf1 and components of the FSH-ovary axis, including plasma FSH, estradiol-17beta, and ovarian mRNAs for gonadotropin receptors, star, igf1, and igf2. A consistent positive relationship between plasma Igf1, estradiol-17beta, and pituitary FSH during growth in the spring suggests that these factors are important links in the mechanism by which body growth influences the rate of oocyte development.

Time course of the GH/IGF axis response to fasting and increased ration in chinook salmon (Oncorhynchus tshawytscha).

Body growth in vertebrates is chiefly regulated by the GH/IGF axis. Pituitary growth hormone (GH) stimulates liver insulin-like growth factor-I (IGF-I) production. During fasting, plasma IGF-I levels decline due to the development of liver GH resistance, while GH levels generally increase. In mammals, decreased insulin during fasting is thought to cause liver GH resistance. However, the sequence of events in the GH/IGF axis response to fasting is not well characterized, especially in non-mammalian vertebrates. We assessed the time course of the GH/IGF axis response to fasting and increased ration in chinook salmon. Fish were placed on Fasting, Increased, or Control rations, and sampled daily for 4 days and at more widely spaced intervals through 29 days. Plasma IGF-I, GH, insulin, and 41 kDa IGF binding protein (putative salmon IGFBP-3), and liver IGF-I gene expression were measured. Control and Increased ration fish did not differ strongly. Plasma IGF-I and 41 kDa IGFBP were significantly lower in Fasted versus Control fish from day 4 onward, and liver IGF-I gene expression was significantly lower from day 6 onward. Liver IGF-I gene expression and plasma IGF-I levels were correlated. Plasma insulin was lower in Fasted fish from day 6 onward. There was a trend toward increased GH in Fasted fish on days 1-2, and GH was significantly increased Fasted fish from day 3 onward. Fasted GH first increased (days 1-3) to a plateau of 10-20 ng/ml (days 4-12) and then increased dramatically (days 15-29), suggesting that the GH response to fasting had three phases. The early increase in GH, followed by the decrease in plasma IGF-I after 4 days, suggests that GH resistance developed within 4 days.

Oestrogen-mediated suppression of tumour necrosis factor alpha-induced apoptosis in MCF-7 cells: subversion of Bcl-2 by anti-oestrogens.

In oestrogen receptor (ER)-positive breast carcinoma cells, 17beta-oestradiol suppresses a dose-dependent induction of cell death by tumour necrosis factor alpha (TNF). The ability of oestrogens to promote cell survival in ER-positive breast carcinoma cells is linked to a coordinate increase in Bcl-2 expression, an effect that is blocked with the pure anti-oestrogen ICI 182,780. The role of Bcl-2 in MCF-7 cell survival was confirmed by stable overexpression of Bcl-2 which resulted in suppression of apoptosis induced by doxorubicin (DOX), paclitaxel (TAX) and TNF as compared to vector-control cells. The pure anti-oestrogen ICI 182,780 in combination with TNF, DOX or TAX potentiated apoptosis in vector-transfected cells. Interestingly, pre-treatment with ICI 182,780 markedly enhanced chemotherapeutic drug- or TNF-induced apoptosis in Bcl-2 expressing cells, an effect that was correlated with ICI 182,780 induced activation of c-Jun N-terminal kinase. Our results suggest that the effects of oestrogens/anti-oestrogens on the regulation of apoptosis may involve coordinate activation of signalling events and Bcl-2 expression.

The effect of low temperature and fasting during the winter on metabolic stores and endocrine physiology (insulin, insulin-like growth factor-I, and thyroxine) of coho salmon, Oncorhynchus kisutch.

The objective of this study was to examine the effect of winter feeding and fasting at both high (10 degrees ) and low (2.5 degrees ) temperatures on growth, metabolic stores, and endocrinology of coho salmon. Treatments were as follows: warm-fed, warm-not fed, cold-fed, and cold-not fed during the winter (January-February). The following parameters were measured: length, weight, whole body lipid, liver glycogen, hepatosomatic index, and plasma levels of insulin, insulin-like growth factor-I (IGF-I), and thyroxine (T4). Warm-fed fish grew continuously throughout the experiment from 21.5 +/- 0.3 to 43.4 +/- 1.4 g and were larger than fish in the other treatments. Fish in all other treatments grew from 21.5 +/- 0.3 to approximately 32 g and showed depressed growth during January and February. During the winter, liver glycogen, hepatosomatic index, plasma insulin, and IGF-I were highly influenced by manipulations in rearing conditions, whereas whole body lipid and plasma T4 were less affected. Plasma insulin levels fluctuated dramatically (from 2 to 7 ng/ml) in the two cold-acclimated groups shortly after the change in temperature. In general, the plasma insulin levels of the warm-fed fish were the highest (8-9 ng/ml), those of the warm-not fed fish were the lowest (2-5 ng/ml), and those of the two cold-acclimated groups were more variable but intermediate. In contrast, plasma IGF-I levels showed a decline with temperature decrease (from 9 to 5 ng/ml) and more gradual changes than insulin with the change in feeding. The highest plasma IGF-I levels were found in the warm-fed fish (10-15 ng/ml), the lowest levels were in the cold-not fed fish (4-5 ng/ml), and those of the warm-not fed and cold-fed fish were intermediate. During the treatment period the T4 levels were relatively unaffected by manipulations in feeding and temperature compared with either insulin or IGF-I. These data suggest that the insulin, IGF-I, and thyroid axes are differentially regulated under changing seasonal and/or environmental conditions in yearling salmon.

NF-kappa B-mediated chemoresistance in breast cancer cells.

BACKGROUND: Nuclear factor-kappa B (NF-kappa B) is a known survival pathway, and it may explain differential sensitivity to tumor necrosis factor-alpha (TNF-alpha) and chemotherapeutic-induced apoptosis in apoptotically sensitive (APO+) and apoptotically resistant (APO-) Michigan Cancer Foundation-7 breast cancer cells. METHODS: Crystal violet viability and luciferase reporter gene assays were used to determine the inhibitory concentration of viability at 50% (IC(50)) and the inhibitory concentration of activity at 50% (EC(50)) values in APO- and APO+ cells with the selective NF-kappa B inhibitor, BAY 11-7082 (BAY). The apoptotic reporter assay was used to determine the effects of the transfection of the inhibitory kappa B-dominant negative (I kappa B-DN) construct in conjunction with TNF, paclitaxel, or doxorubicin treatments in these cells. RESULTS: The concentrations at which 50% of cell viability is inhibited (IC(50)) and at which 50% of NF-kappa B activity is inhibited (EC(50)) for BAY in APO- and APO+ cells were 95.24 micromol/L and 1.53 micromol/L, respectively, and 7.62 micromol/L and 2.64 micromol/L, respectively. The IC(50) and the EC(50) values were equivalent for the APO+ cells (P =.665), but not for the APO- cells (P =.025). I kappa B-DN--transfection alone, or with TNF, doxorubicin, or paclitaxel treatments resulted in cell death of both APO- and APO+ cells as compared with vector-control; however, greater cytotoxicity was seen in the APO+ cells. Direct comparison of the APO+ cells versus the APO- cells revealed that these differences were significant (P =.05). CONCLUSIONS: Pharmacologic or molecular inhibition of the NF-kappa B pathway blocked cell survival in MCF-7 APO+ cells, while only molecular inhibition induced cytotoxicity in the APO- cells. Selective manipulation of the NF-kappa B pathway in combination with standard chemotherapeutic agents may lead to an increased potency and efficacy of these agents.

Gene microarray analysis reveals a novel hypoxia signal transduction pathway in human hepatocellular carcinoma cells.

The molecular details of hypoxia-induced cellular responses have been difficult to identify since there is as yet no known oxygen receptor. We used cDNA microarray technology to extend our studies pertaining to these molecular details in human hepatocellular carcinoma (Hep3B) cells that produce erythropoietin (Epo) in response to hypoxia. Of approximately 1200 genes in the array, those associated with integrin-linked kinase (ILK), fibronectin precursor and glycogen synthase kinase-3beta (GSK-3beta) were markedly stimulated after exposure of Hep3B cells to low oxygen (1%) for 6 h. Epo, HIF-1, and von Hippel-Lindau cDNAs were measured in parallel as markers of low oxygen responses in Hep3B cells. ILK is a serine, threonine protein kinase that interacts with the cytoplasmic domains of integrin beta1 and beta3. This interaction localizes ILK to focal adhesion plaques. ILK is stimulated by cell-fibronectin interaction as well as insulin. It is regulated in a phosphatidylinositol 3-kinase dependent manner and can phosphorylate protein kinase B (PKB/AKT) and GSK-3beta. As a result of these and other activities ILK has been shown to affect anchorage-independent cell survival, cell cycle progression and tumorigenesis in nude mice. ILK has also been implicated in the Wnt pathway and as a critical target in PTEN-dependent tumor therapies. To our knowledge this is the first report implicating the ILK pathway in low oxygen responses. Other genes identified as a result of the microarray analysis not previously known to change as a result of low oxygen treatment were elongation factor-1alpha, glycyl-tRNA synthetase, and laminin receptor protein-1. These findings were all corroborated by RT-PCR assays and in some instances Western blot analysis.

Relationship of insulin-like growth factor-I and insulin to size and adiposity of under-yearling chinook salmon.

Sub-yearling spring chinook salmon were fed either a LoFat or HiFat diet from February to November. Fish were sampled over 2 days in November, following 24- and 48-h fasts. Length vs. weight relationships between fish fed the two diets were similar; however, fish fed the HiFat diet had roughly twice the body lipid as fish fed the LoFat diet (9% vs. 4.5%, respectively). Plasma IGF-I vs. length relations between fish fed the two diets were similar; overall, there was a strong relation between plasma IGF-I and length (r(2)=0.53). Similarly, plasma log (insulin) vs. length relations did not vary between the two diets; however, the relationship of log (insulin) vs. length was weak (r(2)=0.2). There was little or no relationship between plasma IGF-I or log (insulin) and body adiposity. Finally, there was a weak relationship between plasma IGF-I and log (insulin) (r(2)=0.23).

Cobalt induces heme oxygenase-1 expression by a hypoxia-inducible factor-independent mechanism in Chinese hamster ovary cells: regulation by Nrf2 and MafG transcription factors.

We have shown previously that activation of the heme oxygenase-1 (ho-1) gene by hypoxia in aortic smooth muscle cells is mediated by hypoxia-inducible factor-1 (HIF-1). In mutant (Ka13) Chinese hamster ovary cells lacking HIF activity, accumulation of ho-1 mRNA in response to hypoxia and the hypoxia-mimetic CoCl(2) was similar to that observed in wild type (K1) cells. These results support the existence of HIF-dependent and HIF-independent mechanisms for ho-1 gene activation by hypoxia and CoCl(2). In Ka13 cells, CoCl(2) stimulated expression of a luciferase reporter gene under the control of a 15-kilobase pair mouse ho-1 promoter (pHO15luc). Mutation analyses identified the cobalt-responsive sequences as the stress-response elements (StREs). In electrophoretic mobility shift assays, two specific StRE-protein complexes were observed using extracts from Ka13 cells. In response to cobalt, the level of the slower migrating complex X increased, whereas that of complex Y decreased, in a time-dependent manner. Members of the AP-1 superfamily of basic-leucine zipper factors bind to the StRE. Antibody supershift electrophoretic mobility shift assays did not detect Jun, Fos, or ATF/CREB proteins but identified Nrf2 and the small Maf protein, MafG, as components of complex X. Furthermore, dominant-negative mutants of Nrf2 and small Maf, but not of other bZIP factors, attenuated cobalt-mediated gene activation. Additional experiments demonstrated that induction by cobalt does not result from increased expression of MafG or regulated nuclear translocation of Nrf2 but is dependent on cellular oxidative stress. Unlike cobalt, hypoxia did not stimulate pHO15luc expression and did not increase StRE binding activity, indicating distinct mechanisms for ho-1 gene activation by cobalt and hypoxia in Chinese hamster ovary cells.

Effects of ration on somatotropic hormones and growth in coho salmon.

We examined the response of growth hormone (GH), total plasma insulin-like growth-factor I (IGF-I), and growth rate to a change in ration in coho salmon. Tanks of individually tagged fish were placed on high, medium, or low ration, and sampled every 2 weeks for 8 weeks to create a range of growth rates. Some fish received non-lethal blood draws, while others were sampled terminally. Plasma IGF-I levels were higher in high ration fish than in low ration fish from 4 weeks after the beginning of experimental diets to the end of the experiment. GH levels were low and similar in all fish after changing rations, except for the fish in the low ration group at week 2. IGF-I was strongly correlated with specific growth rate in weight in terminally sampled fish after 4 weeks. GH did not correlate with growth rate or IGF-I levels. Growth parameters (length, weight, specific growth rates in weight and length, and condition factor) responded to ration. Serial sampling reduced growth rates and hematocrit, but did not change hormone levels. This study shows that IGF-I responds to changed rations within 2-4 weeks in salmonids.

Comprehension and storage of four serially presented radio news stories by mild aphasic subjects.

The present study investigated aphasic subjects' ability to comprehend and store serially presented discourse. Sixteen mild aphasic subjects, eight age-matched normals, and eight younger normals listened to four serially presented radio news stories and a single radio news story. Half of the aphasic subjects performed as well as age-matched normals in a single-news-story comprehension task. However, they demonstrated a drastic deterioration in performance when asked to listen to a series of four news stories. Age-matched normals, and aphasic subjects, to a lesser extent, showed an impairment in the comprehension and storage of the news story heard last in a series of four news stories. These results were discussed in terms of the comprehension and storage resources of working memory.

PI3-K/AKT regulation of NF-kappaB signaling events in suppression of TNF-induced apoptosis.

We found that in MCF-7 breast carcinoma cells, PI3K and Akt suppressed a dose-dependent induction of apoptosis by tumor necrosis factor alpha (TNF). PI3K and Akt stimulated NF-kappaB activation in a dose-dependent manner, suggesting a common link between these two pathways. TNF has been shown to activate both an apoptotic cascade, as well as a cell survival signal through NF-kappaB. PI3K and AKT cell survival signaling were correlated with increased TNF-stimulated NF-kappaB activity in MCF-7 cells. We demonstrate that while both TNFR1 and NIK are partially involved in Akt-induced NF-kappaB stimulation, a dominant negative IkappaBalpha completely blocked Akt-NF-kappaB cross-talk. PI3K-Akt signaling activated NF-kappaB through both TNFR signaling-dependent and -independent mechanisms, potentially representing a mechanism by which Akt functions to suppress apoptosis in cancer.

Differences in protein kinase C and estrogen receptor alpha, beta expression and signaling correlate with apoptotic sensitivity of MCF-7 breast cancer cell variants.

Widespread use of MCF-7 human breast cancer cells as a model system for breast cancer has lead to variations in these cells between different laboratories. Although several reports have addressed these differences in terms of proliferation and estrogenic response, differences in sensitivity to apoptosis have just begun to be described. Based on the possible differences in apoptotic sensitivity that may arise due to the existence of MCF-7 cell variants, we determined the relative sensitivity of MCF-7 cell variants from three established laboratories (designated M, L and N) to known inducers of apoptosis. Consistent with our previous studies we demonstrate that differences exist among these variants in regards to tumor necrosis factor alpha (TNF)-induced cell death and inhibition of proliferation in a dose-dependent manner. To establish if the difference in apoptotic susceptibility was specific to TNF, the three MCF-7 cell variants were tested for their response to other known inducers of apoptosis: okadaic acid, staurosporine and 4-hydroxy-tamoxifen. Viability and DNA fragmentation analysis revealed a similar pattern of resistance to apoptosis by all agents in the MCF-7 M variant. The MCF-7 L variant was resistant to okadaic acid and 4-hydroxy-tamoxifen but not staurosporine. In contrast, MCF-7 N cells were sensitive to induction of apoptosis by all agents. The role of both protein kinase C (PKC) and estrogen signaling in the regulation of cell survival prompted investigation of these pathways as a mechanism for differential sensitivity of MCF-7 cell variants to apoptosis. While both estrogen receptor alpha (ERalpha) and ERbeta were expressed in MCF-7 M and N cells, the absence of ERbeta in MCF-7 L cells correlated with decreased estrogen responsiveness of the L variant. Variations in estrogenic responsiveness and PKC isoform expression may account for the enhanced susceptibility of both the L and N variants to staurosporine.

Cross-talk between phosphatidylinositol 3-kinase and sphingomyelinase pathways as a mechanism for cell survival/death decisions.

Peptide hormones act to regulate apoptosis through activation of multiple pro- and anti-apoptotic signaling cascades of which lipid signaling events represent an important facet of the cellular rheostat that determines survival and death decisions. Activation of sphingomyelinase, which generates ceramide, is an intermediate in cellular stress responses and induction of apoptosis in many systems. Conversely, phosphatidylinositol 3-kinase (PI3K) is a critical signaling molecule involved in regulating cell survival and proliferation pathways. In the present study, we investigate cross-talk between the PI3K and sphingomyelinase pathways as a mechanism for regulation of cell survival/death decisions. We show that phorbol ester, insulin-like growth factor 1, and a constitutively active PI3K suppress both tumor necrosis factor-induced apoptosis and ceramide generation. Conversely, inhibition of the PI3K pathway with expression of a kinase-dead PI3K both prevented survival signaling and enhanced tumor necrosis factor-induced ceramide generation. The ability of exogenous sphingomyelinase to induce ceramide generation was partially suppressed by expression of constitutively active PI3K and enhanced by inhibition of PI3K suggesting that cross-talk between PI3K and ceramide generation within cells is regulated subsequent to activation of sphingomyelinase.

PH20: a novel tumor marker for laryngeal cancer.

OBJECTIVE: To determine whether levels of PH-20, a hyaluronidase similar to that found in human sperm, are elevated in laryngeal cancer tissue. DESIGN: In this case-control study. reverse transcription polymerase chain reaction was used to measure levels of PH-20 messenger RNA in tissue taken from laryngectomy specimens. SETTING: A university medical center. PATIENTS: We compared tissue samples taken from 11 patients with laryngeal cancer, and from 2 metastatic lymph nodes, with samples of normal, healthy laryngeal tissue and prostate cancer tissue (positive control). MAIN OUTCOME MEASURE: PH-20 complementary DNA expression as quantified by densitometric analysis. RESULTS: Expression of PH-20 was significantly higher in nonirradiated laryngeal cancer specimens than in normal laryngeal tissue (P<.01). Metastatic lymph nodes also had higher levels of PH-20 expression than did primary laryngeal cancer tissue (P = .11) and normal laryngeal tissue (P<.01). Irradiated laryngeal cancer specimens had PH-20 levels comparable to normal. CONCLUSIONS: We report the first data on PH-20 expression in laryngeal cancer tissue. PH-20 expression is significantly elevated in primary laryngeal cancer tissue and seems to be even higher in metastatic lesions compared with normal laryngeal tissue. PH-20 may be a useful tumor marker and prognostic tool for laryngeal cancer.