Biomedical subjects

M Ivan

Publications and source records attributed to M Ivan.

At least 19 recordsLinked to original sources

PubMed2006

Duodenal flow and digestibility in fauna-free sheep and in sheep monofaunated with Entodinium caudatum or Polyplastron multivesiculatum.

Three groups of five rumen and duodenum cannulated fauna-free sheep were used in a 28 d experiment. One group remained fauna-free, whereas the second (EN) and third (PP) groups, respectively, were inoculated intraruminally with the protozoan species Entodinium caudatum and Polyplastron multivesiculatum. Rumen fluid, duodenal digesta and faecal samples were collected during the last 12 d. The flow of digesta to the duodenum was determined using Yb and Co as dual-phase markers. (15)Nitrogen and phosphatidylcholine were used as markers to calculate the duodenal flow of bacterial and protozoal N, respectively. Results showed an increase (P < 0.1) in the rumen concentration of NH3-N and total volatile fatty acids, and a decrease (P < 0.05) in the duodenal flow of non-NH3-N and bacterial N in sheep with EN and PP monofaunas, compared with fauna-free sheep. There were no differences (P > 0.05) in these variables between the two monofauna groups. Protozoal N accounted for 8 % of the duodenal non-NH3-N flow in the EN-monofaunated sheep, whereas no such flow was detected in the PP-monofaunated sheep. Apparent rumen digestibility of organic matter, neutral detergent fibre and acid detergent fibre were similar (P > 0.05) in the monofaunated groups of sheep, but rumen acid detergent fibre digestibility was higher (P < 0.05) in the monofaunated than in the fauna-free groups. Experimental results suggested that, unlike EN, the PP monofauna might not contribute to the duodenal flow of microbial protein, whereas both monofaunas showed a virtually equal degree of predation on rumen bacteria.

Animal Feed↗

PubMed2005

Effects of flaxseed on protein requirements and N excretion of dairy cows fed diets with two protein concentrations.

Thirty-eight midlactating Holstein cows averaging 597 kg of body weight (SD = 59) were used to determine the effects of dietary flaxseed on protein requirement and N excretion in urine and feces. Milk yield and composition, intake, and digestibility were also determined. Cows were allotted from wk 20 to 30 of lactation to 1 of 4 TMR containing 1) no flaxseed (control) and 16% protein (MPC), 2) whole flaxseed and 16% protein (MPF), 3) no flaxseed (control) and 18% protein (HPC), and 4) whole flaxseed and 18% protein (HPF). Cows fed high protein diets had greater feed intake than those fed medium protein diets (20.2 vs. 18.4 kg/d), and cows fed no flaxseed had greater dry matter intake than those fed flaxseed (20.1 vs. 18.5 kg/d). Milk yield was lower for cows fed MPF (20.3 kg/d) than for those fed HPC (24.4 kg/d), HPF (24.9 kg/d), or MPC (24.0 kg/d). Milk protein and lactose concentrations were similar for cows fed MPC and HPC, but flaxseed decreased milk protein concentration in cows fed MPF or HPF compared with cows fed the control diets. Milk fat concentration was similar in cows fed diets with or without flaxseed, but it was decreased by higher protein concentration. Digestibility was generally reduced when diets contained flaxseed and lower protein concentration. Dietary protein had no effect while dietary flaxseed increased fecal N excretion. Retention of N was lower in cows fed flaxseed compared with cows fed the control diets. Feeding flaxseed decreased milk concentrations of short- and medium-chain fatty acids and increased those of long-chain fatty acids. Flaxseed had no effect on the dietary requirement of N by midlactating dairy cows.

Animal Nutritional Physiological Phenomena↗

PubMed2004

Effects of dietary sunflower seeds on rumen protozoa and growth of lambs.

Linoleic acid-rich sunflower-seed supplements (SSS) were used in two experiments (experiment 1, high-concentrate diets; experiment 2, high-forage diets) to study effects on rumen protozoa and the growth of lambs. Both experiments consisted of four treatments, two with a low-protein diet (120 g/kg) and two with a high-protein diet (160 g/kg). For both diets, one treatment was without (control) and one with the SSS (140 g/kg dietary DM). The lambs were fed ad libitum for 70 and 140 d in experiments 1 and 2, respectively. Thereafter, the digestibility of organic matter (OM), acid-detergent fibre and neutral-detergent fibre were determined for each diet with four lambs, and then all lambs were slaughtered and rumen fluid samples were collected and analysed. The results showed substantial decreases (P < 0.001) or total elimination of protozoa in the rumen fluid of the SSS-receiving lambs. In the first experiment the SSS also decreased (P < 0.05) feed intake, but an increase in average daily gain (P < 0.06) resulted in an improved (P < 0.05) feed:gain ratio. Also, the SSS increased (P < 0.05) the digestibility of fibre. In the second experiment the SSS decreased (P < 0.05) the OM digestibility, feed intake and growth of lambs. It was concluded that the use of sunflower-seed supplementation in high-concentrate diets of ruminants reduces rumen fauna, resulting in savings on dietary protein supplements and an increased digestion of feed.

Animals↗

PubMed2004

Value of sunflower seed in finishing diets of feedlot cattle.

The value of sunflower seed (SS) in finishing diets was assessed in two feeding trials. In Exp. 1, 60 yearling steers (479 +/- 45 kg) were fed five diets (n = 12). A basal diet (DM basis) of 84.5% steam-rolled barley, 9% barley silage, and 6.5% supplement was fed as is (control), with all the silage replaced (DM basis) with rolled SS, or with grain:silage mix replaced with 9% whole SS, 14% whole SS, or 14% rolled SS. Liver, diaphragm, and brisket samples were obtained from each carcass. In Exp. 2, 120 yearling steers (354 +/- 25 kg) were fed corn- or barley-based diets containing no SS, high-linoleic acid SS, or high-oleic acid SS (a 2 x 3 factorial arrangement, n = 20). Whole SS was included at 10.8% in the corn-based and 14% in the barley-based diets (DM basis). In Exp. 1, feeding whole SS linearly increased DMI (P = 0.02), ADG (P = 0.01), and G:F (P = 0.01). Regression of ME against level of whole SS indicated that SS contained 4.4 to 5.9 Mcal ME/kg. Substituting whole for rolled SS did not significantly alter DMI, ADG, or G:F (8.55 vs. 8.30 kg/d; 1.36 vs. 1.31 kg; and 0.157 vs. 0.158, respectively). Replacing the silage with rolled SS had no effect on DMI (P = 0.91) but marginally enhanced ADG (P = 0.10) and improved G:F (P = 0.01). Dressing percent increased linearly (P = 0.08) with level of SS in the diet. Feeding SS decreased (P < 0.05) levels of 16:0 and 18:3 in both diaphragm and subcutaneous fats, and increased (P = 0.05) the prevalence of 18:1, 18:2, cis-9,trans-11-CLA and trans-10,cis-12-CLA in subcutaneous fat. In Exp. 2, barley diets supplemented with high-linoleic SS decreased DMI (P = 0.02) and ADG (P = 0.007) by steers throughout the trial, whereas no decrease was noted with corn (interaction P = 0.06 for DMI and P = 0.01 for ADG). With barley, high-linoleic SS decreased final live weight (554 vs. 592 kg; P = 0.01), carcass weight (329 vs. 346 kg; P = 0.06), and dressing percent (58.5 vs. 59.4%; P = 0.04). Steers fed high-linoleic SS plus barley had less (P < 0.05) backfat than those fed other SS diets. No adverse effects of SS on liver abscess incidence or meat quality were detected. Although they provide protein and fiber useful in formulating finishing diets for cattle, and did improve performance in Exp. 1, no benefit from substituting SS for grain and roughage was detected in Exp. 2. Because of unexplained inconsistencies between the two experiments, additional research is warranted to confirm the feeding value of SS in diets for feedlot cattle.

Adipose Tissue↗

PubMed2004

In vitro effects of individual fatty acids on protozoal numbers and on fermentation products in ruminal fluid from cattle fed a high-concentrate, barley-based diet.

The objective of this study was to investigate the effects of sodium salts of individual fatty acids on protozoal numbers and ruminal fermentation variables in vitro. Ruminal inoculum was obtained from two heifers fed a finishing diet consisting of (DM basis) 90% rolled barley grain, 4% barley silage, 5% soybean meal, and 1% mineralized salt. Fatty acids (FA) were included individually in the inoculum as follows: C6:0, C8:0, and C10:0 at concentrations (wt/vol) of 0.0625, 0.125, and 0.25%; C14:0 and C18:0 at concentrations of 0.125, 0.25, and 0.5%; and C12:0, C16:0, C18:1, C18:2, and C18:3 at concentrations of 0.25, 0.5, and 1.0%. 15N-Labeled casein was included as a N tracer. In the presence of medium-chain saturated FA (particularly C10:0 and C12:0), no ciliate protozoa (99.8%Entodinium spp.) were recovered from the incubation medium. Long-chain unsaturated FA (C18:3, C18:2, C18:1) also decreased (P < 0.05) protozoal numbers. At all concentrations tested, C10:0 and C12:0 decreased (P < 0.05) ammonia and total VFA concentrations (by 29 and 22%, respectively) and increased (P < 0.05) concentrations of total free amino acids, reducing sugars, and soluble protein. At the greatest concentrations of these FA, xylanase and amylase activities of the incubation media were decreased (P < 0.05). The C18 unsaturated FA increased (P < 0.05) the polysaccharide-degrading activities of the media. These in vitro results suggest that long-chain unsaturated FA in combination with medium-chain saturated acids have the potential to decrease protozoal numbers and ruminal ammonia utilization in cattle fed high-grain diets.

Ammonia↗

PubMed2001

von Hippel-Lindau protein mutants linked to type 2C VHL disease preserve the ability to downregulate HIF.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germ line mutation of the von Hippel-Lindau tumor suppressor gene (VHL). Tumors observed in this disorder include retinal and central nervous system hemangioblastomas, clear cell renal carcinomas and pheochromocytomas. The VHL gene product, pVHL, is a component of a ubiquitin ligase which targets the transcription factor known as hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. pVHL also plays roles in the control of extracellular matrix formation and cell-cycle exit. Different VHL mutations confer different site-specific risks of cancer. Type 2C VHL mutations confer an increased risk of pheochromocytoma without the other stigmata of VHL disease. Here we report that the products of such type 2C VHL alleles retain the ability to down regulate HIF but are defective for promotion of fibronectin matrix assembly. Furthermore, pVHL L188V, a well studied type 2C mutant, retained the ability to suppress renal carcinoma growth in vivo. These studies strengthen the notion that HIF deregulation plays a causal role in hemangioblastoma and renal carcinoma, and raises the possibility that abnormal fibronectin matrix assembly contributes to pheochromocytoma pathogenesis in the setting of VHL disease.

Alleles↗

PubMed2001

HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.

HIF (hypoxia-inducible factor) is a transcription factor that plays a pivotal role in cellular adaptation to changes in oxygen availability. In the presence of oxygen, HIF is targeted for destruction by an E3 ubiquitin ligase containing the von Hippel-Lindau tumor suppressor protein (pVHL). We found that human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. Because proline hydroxylation requires molecular oxygen and Fe(2+), this protein modification may play a key role in mammalian oxygen sensing.

Amino Acid Sequence↗

PubMed2001

The von Hippel-Lindau tumor suppressor protein.

The von Hippel-Lindau tumor suppressor protein (pVHL) has been shown to bind directly to the alpha subunits of the heterodimeric transcription factor HIF (hypoxia inducible factor). pVHL directs the polyubiquitination and, hence, destruction of HIF in the presence of oxygen. Loss of pVHL function leads to deregulation of HIF target genes, which play critical roles in angiogenesis.

Animals↗

PubMed2001

Fermentation characteristics and ruminal ciliate protozoal populations in cattle fed medium- or high-concentrate barley-based diets.

Fermentation characteristics were measured and numbers and distribution by genera of ciliate protozoa were determined in ruminal fluid samples collected from 10 ruminally cannulated steers during the first 30 d of their being fed barley-based diets containing 62% (Medium Barley) or 95% (High Barley) barley grain (DM basis). Ruminal samples were collected at 5-d intervals over the 30-d periods beginning after adaptation (i.e., at the first full feeding of each diet). Ruminal pH and ammonia concentrations were lower (P < 0.001) with the High Barley than with the Medium Barley diet. Concentrations of total VFA and propionate and amylase activity of ruminal fluid were higher (P < 0.001) on High Barley than on Medium Barley. Total protozoal numbers in ruminal fluid were 42% lower (P < 0.05) on High Barley (470 x 10(3)/mL) than on Medium Barley (804 x 10(3)/mL). On Medium and High Barley diets, respectively, Entodinium spp. made up 89 and 91% of the ciliate protozoal populations. With the Medium Barley diet, relative proportions of Dasytricha, Ophryoscolex, Ostracodinium, Diplodinium, and Metadinium spp. in the total ciliate population were 4.5, 0.4, 0.5, 0.7, and 0.3%, respectively. When the High Barley diet was fed, these genera were not detected. In a subsequent survey, ruminal samples were collected from 200 finishing cattle at slaughter. Average protozoal population was 328 x 10(3)/mL, and Entodinium spp. constituted 97% of the total. These data demonstrate that a large population of Entodinium spp. can persist in the rumen of cattle fed high barley grain-based finishing diets.

Amino Acids↗

PubMed2000

Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is characterized by the development of multiple vascular tumors and is caused by inactivation of the von Hippel-Lindau protein (pVHL). Here we show that pVHL, through its beta-domain, binds directly to hypoxia-inducible factor (HIF), thereby targeting HIF for ubiquitination in an alpha-domain-dependent manner. This is the first function to be ascribed to the pVHL beta-domain. Furthermore, we provide the first direct evidence that pVHL has a function analogous to that of an F-box protein, namely, to recruit substrates to a ubiquitination machine. These results strengthen the link between overaccumulation of HIF and development of VHL disease.

Cell Extracts↗

PubMed2000

Evidence for a telomere-independent "clock" limiting RAS oncogene-driven proliferation of human thyroid epithelial cells.

An initiating role for RAS oncogene mutation in several epithelial cancers is supported by its high incidence in early-stage tumors and its ability to induce proliferation in the corresponding normal cells in vitro. Using retroviral transduction of thyroid epithelial cells as a model we ask here: (i) how mutant RAS can induce long-term proliferation in an epithelial cell in contrast to the premature senescence observed in fibroblasts; and (ii) what is the "clock" which eventually triggers spontaneous growth arrest even in epithelial clones generated by mutant RAS. The early response to RAS activation in thyroid epithelial cells showed two features not seen in fibroblasts: (i) a marked decrease in expression of the cyclin-dependent kinase inhibitor (CDKI) p27(kip1) and (ii) the absence of any induction of p21(waf1). When proliferation eventually ceased (after up to 20 population doublings) this occurred despite undiminished expression of mutant RAS and was tightly correlated with a return to the initial high level of p27(kip1) expression, together with the de novo appearance of p16(ink4a). Importantly, neither the CDKI changes nor the proliferative life span of RAS-induced epithelial clones was altered by induction of telomerase activity through forced expression of the catalytic subunit, hTERT, at levels sufficient to immortalize human fibroblasts. These data provide a basis for cell-type differences in sensitivity to RAS-induced proliferation which may explain the corresponding tumor-type specificity of RAS mutation. They also show for the first time in a primary human cell model that a telomere-independent mechanism can limit not only physiological but also oncogene-driven proliferation, pointing therefore to a tumour suppressor mechanism additional, or alternative, to the telomere clock.

Catalytic Domain↗

PubMed2000

Ruminal fermentation and duodenal flow following progressive inoculations of fauna-free wethers with major individual species of ciliate protozoa or total fauna.

Naturally fauna-free (FF) wethers, equipped with ruminal and duodenal cannulas, were used in two groups of eight (Group A) and seven (Group B) animals in six consecutive experimental periods, each lasting for 28 d. The objective was to measure ruminal fermentation traits, and flows of nonammonia nitrogen (NAN), total amino acid (TAA), and bacterial nitrogen (BN) from the stomach after inoculation with individual ciliate protozoa species in each period. The wethers in both groups were fed a diet based on corn silage, haylage, and soybean meal, and they remained FF during the first period. At the beginning of each other period, the wethers were progressively inoculated intraruminally with one individual major species of ruminal ciliate protozoa or total fauna (TF). Thus, Group A was progressively inoculated (+) with Dasytricha ruminantium (DS), Polyplastron multivesiculatum (PP), Isotricha intestinalis (IS), Entodinium caudatum (EN) and TF-type A. Also, Group B was progressively inoculated (+) with IS, DS, Epidinium ecaudatum (EP), Eudiplodinium maggi (EU), and EN. Duodenal digesta and ruminal fluid were collected and sampled in each period on d 26 and 28, respectively, and subjected to chemical analyses. A significantly higher (P < .05) pH (6.4) in ruminal fluid of the Group A wethers was obtained when each DS, DS+PP, DS-PP-IS+EN, and TF population was present in the rumen than when the wethers were FF (6.2). In the Group B wethers, pH (6.1) was lower (P < .05) for the population of IS-DS-EP+EU than for other populations (6.2 to 6.3). The concentration of total VFA in ruminal fluid was higher (P < .05) in the Group B wethers when IS, IS+DS, or IS-DS+EP populations were present in the rumen than when the wethers were FF. The flow of NAN, TAA, and BN from the stomach to the intestinal tract was generally lower for different protozoa populations than for the FF period. Largest decreases (P < .05) in the flow of NAN, TAA, and BN occurred when EN was added into the rumen of wethers in the A and B groups, which already contained populations of DS-PP+IS and IS-DS-EP+EU, respectively. Holotrich protozoa had very little effect on the protein metabolism in the rumen, but cellulolytic protozoa (PP, EP, and EU) and EN decreased the efficiency of protein utilization by the ruminant host.

Amino Acids↗

PubMed2000

Effects of Isotricha, Dasytricha, Entodinium, and total fauna on ruminal fermentation and duodenal flow in wethers fed different diets.

The objective was to measure rumen fermentation and duodenal flow of amino acids and nonammonia N components in five groups of five ruminally and duodenally cannulated wethers that were fauna-free, or inoculated with the ciliate protozoa genera of Isotricha, Dasytricha, Entodinium, or a normal population (total) of fauna. They were used in two 25-d periods and fed a haycrop-based diet in the first period and a corn silage-based diet in the second period. Feces, duodenal digesta, and rumen contents were sampled in each period and analyzed. The number of Entodinium in wethers containing the Entodinium monofauna was higher than the total protozoa numbers, including Entodinium, in wethers containing total fauna population. The type of diet or fauna did not affect total volatile fatty acid concentrations in rumen fluid. The ammonia N concentration in rumen fluid was higher in wethers containing total fauna (25 mg/100 ml) than in fauna-free wethers fed the two diets (18 and 12 mg/ 100 ml). In comparison with the respective fauna-free wethers, the concentration of ammonia in wethers containing Entodinium was higher when fed the corn silage diet, but not different when fed the haycrop diet. Ruminal presence of total fauna or Entodinium decreased the nonammonia N by 16 and 17%, and total amino acid flows from the stomach by 20 and 19%, respectively. Flow of bacteria N was decreased in wethers fed the two diets when Entodinium or total fauna were present in the rumen. The presence of Isotricha resulted in higher flow of bacteria N in wethers fed the haycrop diet, but the presence of Dasytricha resulted in higher bacteria N flow in wethers fed the corn silage diet. Entodinium was the most detrimental of ciliate protozoa species concerning protein nutrition of the host ruminant.

Amino Acids↗

PubMed1999

Contrasting effects of activating mutations of GalphaS and the thyrotropin receptor on proliferation and differentiation of thyroid follicular cells.

The cyclic AMP pathway is a major regulator of thyrocyte function and proliferation and, predictably, its inappropriate activation is associated with a sub-set of human thyroid tumours. Activating mutations are, however, more common in the thyrotropin receptor (TSHR) than in its downstream transducer, Galphas. To investigate whether this reflects an inherent difference in their oncogenic potency, we compared the effects of retrovirally-transduced mutant (A623I) TSHR or (Q227L) Galphas (GSP), using the rat thyroid cell line FRTL5 and primary human thyrocytes. In FRTL5, expression of GSP or mutant (m) TSHR induced a 2 - 3-fold increase in basal levels of cAMP. This was associated with TSH-independent proliferation (assessed by both cell number and DNA synthesis) and function (as shown by increased expression of thyroglobulin (Tg) and the sodium/iodide symporter). In primary cultures, expression of mTSHR, but not GSP, consistently induced formation of colonies with epithelial morphology and thyroglobulin expression, capable of 10 - 15 population doublings (PD) compared to less than three in controls. Thus, while mTSHR and GSP exert similar effects in FRTL5, use of primary cultures reveals a major difference in their ability to induce sustained proliferation in normal human thyrocytes, and provides the first direct evidence that mTSHR is sufficient to initiate thyroid tumorigenesis.

Animals↗

PubMed1999

The difference between p53 mutation frequency in haematological and non-haematological malignancies: possible explanations.

p53 gene mutations are the most frequent alterations in human cancers. In the published literature, a highly significant statistical difference between the prevalence of p53 mutations in haematological (H) and non-haematological (NH) malignancies can be found. However, no consistent reasons have been suggested to explain it. We propose two non-exclusive possibilities: (i) in H tumours p53 is altered with the same frequency as in NH tumours, but mechanisms other than mutations are involved and (ii) in H malignancies the prevalence of p53 mutations is much lower than in NH cancers because other genetic disturbances are involved. We hypothesized that retention of wild-type p53 in H cancers may be a consequence of: (a) the presence of telomerase activity in haematological cells and/or (b) the absence of hypoxia in the majority of H tumours.

Hematologic Neoplasms↗

PubMed1997

Activated ras and ret oncogenes induce over-expression of c-met (hepatocyte growth factor receptor) in human thyroid epithelial cells.

Hepatocyte Growth Factor (HGF) receptor, encoded by the protooncogene c-met, is overexpressed in many human tumours, including those of thyroid epithelium. The absence in most cases of any primary structural abnormality of the met gene suggests that overexpression is secondary to mutation of other gene(s). To test this hypothesis we investigated the effect on met expression of two activated oncogenes known to play a major role in thyroid oncogenesis, ras and ret. To minimize the possibility of unknown co-operating events, we introduced these genes directly into normal human thyrocytes in primary culture using amphotropic retroviral vectors and assessed met expression as early as possible in the resulting epithelial colonies. Double immunofluorescence revealed expression of met protein, strictly localized to cells expressing the mutant ras and ret vectors, expression in background normal cells being barely detectable. In contrast, colonies induced to proliferate at a comparable rate by a vector expressing SV40 T showed no increase in met expression. To permit quantitation by Western blotting we also extended these findings to a thyroid cell line (R18) containing a zinc-inducible mutant ras gene. Induction of the oncogene led to a fourfold increase in met protein expression. We conclude that overexpression of met is induced by activation of the ras or ret signalling pathway and not simply by deregulation of the cell cycle per se. The data suggest that the proliferative advantage conferred by these oncogenes may be in part due to the resulting sensitization of tumour epithelium to paracrine HGF secreted by stromal cells.

Antigens, Viral, Tumor↗

PubMed1997

GH3 cells expressing constitutively active Gs alpha (Q227L) show enhanced hormone secretion and proliferation.

cAMP levels in GH3gsp cells (Q227L mutation of Gs alpha), in comparison with uninfected GH3 and GH3vt (with vector alone) cells, were two to three fold (P< 0.01) higher (basal), and 10-20 fold (P<0.001) higher (in the presence of isobutyl methylxanthine, (IBMX)). Proliferation of GH3gsp cells after 7 days in culture, as determined by cell number and [(3)H]thymidine incorporation, were up to 25% (respectively P <0.001 and P < 0.02) higher. After chronic (4 days) but not acute (15 min) exposure to forskolin (10 microM) or dibutyryl cAMP (50 microM) all cell types showed a greater than 200% (P < 0.001) increase in [(3)H]thymidine incorporation. Secretion of prolactin and growth hormone by GH3gsp cells were two to four fold (P <0.001) higher than GH3 and GH3vt cells after 4 h and 10-12 fold (P <0.001) higher after 8 h. In conclusion GH3 cells possessing Q227L have a higher proliferation rate and secrete higher levels of prolactin and growth hormone which are associated with higher levels of cAMP.

1-Methyl-3-isobutylxanthine↗

PubMed1997

An amphotropic retroviral vector expressing a mutant gsp oncogene: effects on human thyroid cells in vitro.

Point mutations of the gsp protooncogene (encoding the alpha-subunit of the Gs protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin-tagged Gln227-->Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA-tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 10(5) infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.

Animals↗