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Biomedical subjects

W W Souba

Publications and source records attributed to W W Souba.

At least 19 recordsLinked to original sources
PubMed1998

Determinants of glutamine dependence and utilization by normal and tumor-derived breast cell lines.

A continual supply of the amino acid glutamine (GLN) may be necessary for cancerous cell growth. GLN plays a central role in multiple metabolic pathways and has long been considered an essential component of tissue culture media. However, the GLN requirements of tumor cell lines and the factors that determine a cell's need for GLN have not been comprehensively studied. Also, it remains unclear how various metabolic pathways contribute to GLN consumption. In the present study, possible determinants of GLN metabolism were examined in seven breast cell lines, two derived from immortalized normal tissue and five of tumor origin. These cells exhibited different dependencies on media GLN concentration for growth and a wide range of GLN utilization rates. GLN uptake was facilitated by a single, common transporter functionally defined as System ASC. However, the affinities for GLN exhibited by this transporter differed appreciably between cell lines. Furthermore, the concentration at which media GLN became a limiting factor for cellular proliferation correlated with transporter affinity. The origin of the cell lines was not a determinant of GLN metabolism because immortalized cells of nontumor origin exhibited GLN dependence and utilization rates comparable to those of tumor-derived cells. The rates of CO2 production from GLN were similar for each cell lines. Rates of GLN disappearance and glutamate appearance in media were strongly correlated, with 32-80% of media GLN converted to glutamate. Both rates were directly affected by media cystine concentration, suggesting that a large portion of glutamate efflux was coupled with cystine import through the amino acid transport system x(c)-. These results demonstrated that cell growth is a function of GLN influx and suggest that GLN is used to supply glutamate and cystine, perhaps for glutathione synthesis.

Biological Transport↗
PubMed1998

Effects of endotoxin challenge on hepatic amino acid transport during cancer.

BACKGROUND: The hepatic uptake of amino acids is increased in both sepsis and cancer, and this response appears to be both global and essential in the catabolic host. Because immunocompromised cancer patients are susceptible to episodes of gram-negative sepsis, we examined the capacity of hepatocytes from normal and tumor-influenced livers to respond to the additional challenge of endotoxemia via increases in the Na+-dependent uptake of glutamine and zwitterionic amino acids by System N and System A, respectively. MATERIALS AND METHODS: Fischer 344 rats were implanted with methylcholanthrene-induced fibrosarcomas. Control rats were sham-operated and pair-fed. Animal pairs (tumor burden = 8-32% carcass weight) were injected intraperitoneally with either Escherichia coli endotoxin (10 mg/kg) or PBS, and after 4 h, hepatocytes were isolated from the livers of the animals via collagenase perfusion and placed in primary culture. Three hours later, amino acid transport rates were measured using radiolabeled glutamine for System N and alpha-methylaminoisobutyric acid (MeAIB), a nonmetabolizable substrate specific for System A. RESULTS: Cancer-independent of tumor size-and endotoxin each elicited similar 1.5- to 2-fold inductions of System N activity. When combined, their effects were additive rather than synergistic. In contrast, endotoxin induced an insignificant increase in System A activity, whereas cancer stimulated this carrier 2-fold in either the absence or the presence of endotoxin. CONCLUSIONS: The primary glutamine and alanine carriers in hepatocytes are differentially influenced during catabolic states, and the tumor-influenced liver is competent to further increase glutamine uptake in response to additional catabolic insults.

Amino Acids↗
PubMed1998

Stimulation of rat hepatic amino acid transport by burn injury.

Burn injury accelerates hepatic amino acid metabolism, but the role of transmembrane substrate delivery in this response has not been investigated. We therefore studied the effects of cutaneous scald injury on the Na+-dependent transport of glutamine and alanine in isolated rat liver plasma membrane vesicles. Scald injury resulted in liver damage and a 1.4- to 2.3-fold and 1.5- to 2.8-fold stimulation of hepatic transport rates for glutamine and alanine, respectively, proportional to the total burned surface area (TBSA) after 24 hours. Enhanced uptake of glutamine and alanine was attributable to increases in the maximum velocity (Vmax) of system N and system A activities, respectively. Hepatic amino acid transport activity remained elevated in vesicles from burned animals after 72 hours, but the degree of stimulation (1.3- to 1.7-fold for glutamine and 1.3- to 1.6-fold for alanine) was less than that observed 24 hours after thermal injury. Liver function tests returned to control values after 72 hours as well, indicating rectification of hepatic damage. In contrast to the induction of hepatic system A and system N activity in catabolic states such as cancer and endotoxemia, further studies showed that tumor necrosis factor (TNF) failed to play a significant role in burn-stimulated amino acid transport rates. When combined with plasma liver enzyme profiles, early transient hepatic amino acid transporter stimulation may support amino acid-dependent pathways involved in the repair of burn-dependent hepatic damage.

Amino Acids↗
PubMed1998

Adaptive alterations in cellular metabolism with malignant transformation.

OBJECTIVE: The authors studied the differences between glutamine and glucose utilization in normal fibroblasts and in fibrosarcoma cells to gain insights into the metabolic changes that may occur during malignant transformation. SUMMARY BACKGROUND DATA: The process of malignant transformation requires that cells acquire and use nutrients efficiently for energy, protein synthesis, and cell division. The two major sources of energy for cancer cells are glucose and glutamine. Glutamine is also essential for protein and DNA biosynthesis. We studied glucose and glutamine metabolism in normal and malignant fibroblasts. METHODS: Studies were done in normal rat kidney fibroblasts and in rat fibrosarcoma cells. We measured glutamine transport across the cell membrane, breakdown of glutamine by the enzyme glutaminase (the first step in oxidation), glutamine and glucose oxidation rates to CO2, rates of protein synthesis from glutamine, and glutamine-dependent growth rates. RESULTS: Glutamine transport rates were increased more than sixfold in fibrosarcomas compared to normal fibroblasts. In fibroblasts, glutamine transport was mediated by systems ASC and A. In malignant fibrosarcomas, only system ASC was identifiable, and its Vmax was 15 times higher than that observed in fibroblasts. Despite an increase in transport, glutaminase activity was diminished and glutamine oxidation to CO2 was reduced in fibrosarcomas versus normal fibroblasts. In fibroblasts, glutamine oxidation was 1.8 times higher than glucose oxidation. In contrast, glucose oxidation was 3.5 times greater than glutamine oxidation in fibrosarcomas. Protein synthesis from glutamine transported by fibrosarcomas was threefold greater than that observed in normal fibroblasts. Despite marked increases in glutamine utilization and glucose oxidation in fibrosarcoma cells, growth rates were higher in the normal fibroblasts. CONCLUSIONS: The process of malignant transformation is associated with a marked increase in cellular glutamine transport, which is mediated by a single high-affinity, high-capacity plasma membrane carrier protein. In normal fibroblasts, the transported glutamine is used primarily for energy production via oxidation of glutamine carbons to CO2. In fibrosarcomas, glutamine oxidation falls and glutamine is shunted into protein synthesis; simultaneously, the malignant cell switches to a glucose oxidizer. The increased glutamine transport and glucose oxidation in fibrosarcomas appears to be related to the malignant phenotype and not merely to an increase in cell growth rates.

Adaptation, Physiological↗
PubMed1998

Multiwell 14CO2-capture assay for evaluation of substrate oxidation rates of cells in culture.

14CO2 capture is commonly used to evaluate the cellular oxidation rate of respiratory substrates. A modification of the established 14CO2-capture method was developed that enables the use of cells in adherent culture and easy analysis of multiple samples under different culture conditions. The use of commercially available culture and filter plates designed for use in a multiplate scintillation spectrophotometer enabled substrate oxidation rates to be evaluated for cells in a 24-well plate format without the need to dislodge the cells from the culture substrate as is required in traditional methods. Evaluation of radioactivity captured in potassium hydroxide-saturated filters was accomplished by adding scintillation fluid to the filter plate wells and counting. Alternatively, filters could be removed and placed in vials for evaluation in a conventional scintillation counter. This method was applied to the oxidation of 14C-glutamine by human breast cell lines and demonstrated concentration-dependent linear accumulation of captured counts.

Breast Neoplasms↗
PubMed1998

Extrinsic innervation modulates canine jejunal transport of glutamine, alanine, leucine, and glucose.

BACKGROUND: We previously showed a decrease in ileal glutamine transport in vitro and net absorption in vivo after extrinsic denervation of the canine jejunoileum. The aim was to determine whether extrinsic innervation modulates in vivo net absorption and in vitro transport of glutamine and other nutrients in canine jejunum. METHODS: In vivo net jejunal uptakes of glutamine, alanine, leucine, and glucose were measured in five dogs before and 2 and 8 weeks after a model neurally isolating in situ the jejunoileum (extrinsic denervation, intestinal transection). To assess mechanisms, carrier-mediated uptakes were quantitated in jejunal brush border membrane vesicles from six dogs before and at 2 and 8 weeks after neural isolation of the jejunoileum and compared with six control dogs with fully intact extrinsic innervation. RESULTS: In vivo net absorption of glutamine decreased at 2 weeks (p < 0.05) and returned to normal values at 8 weeks; net absorptions of leucine, alanine, and glucose were decreased at both 2 and 8 weeks. In vitro brush border membrane vesicles transport of glutamine, leucine, and alanine followed the patterns of in vivo absorption, but glucose transport did not differ at any time point. Decreased glutamine uptake at 2 weeks resulted from a decrease in Vmax rather than a change in K(m) in sodium-dependent carrier-mediated transport. CONCLUSIONS: Extrinsic denervation down-regulated carrier-mediated transport of amino acids but not glucose. Decreased in vitro glutamine transport was mediated in part by a decrease in number rather than affinity of sodium-dependent transporters.

Alanine↗
PubMed1998

Protein kinase C regulates nutrient uptake and growth in hepatoma cells.

BACKGROUND: Human hepatoma cells extract glutamine at rates severalfold greater than normal hepatocytes through a high-affinity transporter encoded by the ATB0 gene, which contains two putative phosphorylation sites for protein kinase C (PKC). The studies presented here were undertaken to determine whether System B0-mediated glutamine uptake regulates hepatoma growth and whether PKC regulates the activity of this transporter. METHODS: SK-Hep cells were treated with the PKC activator phorbol 12-myristate 13-acetate (PMA) and the initial-rate transport of glutamine and other nutrients measured at specific times thereafter. Growth rates were monitored during culture +/- PMA or an excess of system B0 substrates relative to glutamine. RESULTS: PMA treatment exerted a rapid (half-life approximately 15 minutes) concentration-dependent inhibition of glutamine uptake rates to 50% of control values via a posttranslational mechanism that decreased transporter maximum velocity. This effect persisted after 24 hours and was abrogated by the PKC inhibitor staurosporine. PMA also significantly decreased amino acid transport System y+ and System L activities but no System A. Chronic treatment with PMA (PKC depletion) inhibited SK-Hep growth, as did attenuation of System B0-mediated glutamine uptake with other B0 substrates. CONCLUSIONS: System B0-mediated glutamine uptake regulates hepatoma cell growth, whereas PKC influences both processes.

Amino Acid Transport Systems↗
PubMed1997

Hepatic glutaminase gene expression in the tumor-bearing rat.

UNLABELLED: Previous studies have documented an increase in hepatic plasma membrane glutamine transport in the tumor-bearing rat, but the effects of tumor burden on hepatic glutaminase expression have not been carefully studied. The purpose of this study was to examine the effects of tumor burden and food intake on hepatic glutaminase expression. Rats were implanted with syngeneic methylcholanthrene-induced fibrosarcoma tumor tissue; control rats were sham operated and pair-fed every 24 hr. Northern blotting was used to assay the effect of tumor burden and fasting on hepatic glutaminase mRNA levels, using beta-actin mRNA as a control. Hepatic glutaminase mRNA levels in livers of pair-fed controls were found to be 4-fold greater than levels in livers of tumor-bearing animals. Examination of food intake patterns in these animals indicated that pair-fed controls ate their allotted chow quickly while tumor-bearing rats ate small amounts throughout each 24 hr period. This observation suggested that the differences in glutaminase mRNA levels may be due to a period of fasting by pair-fed animals which was not experienced by the tumor-bearing group. Hepatic glutaminase mRNA levels rapidly increased in normal rats during acute fasting to levels 5.5-fold greater than fed animals. Glucose feeding and insulin injection rapidly reversed the effect of fasting on hepatic glutaminase mRNA levels in normal rats. Tumor-bearing rats also exhibited upregulation of hepatic glutaminase mRNA levels in response to fasting. CONCLUSIONS: (1) Tumor burden itself does not alter hepatic glutaminase expression, at least at the pre-translational level. Instead, differences in hepatic glutaminase mRNA content are due to differences in food intake patterns. (2) Hepatic glutaminase mRNA levels are rapidly upregulated in response to fasting, an effect which appears to be linked to a decrease in plasma insulin concentrations. Because tumor-bearing rats eat regularly over a 24 hr period (albeit in small increments), thereby maintaining the plasma insulin concentration, hepatic glutaminase mRNA may not rise as it does in pair-fed controls whose daily chow intake is complete within hours of food allocation. (3) This study indicates that differences in the timing of food intake between tumor-bearing rats and pair-fed controls can alter the expression of genes that are influenced by nutrient availability. These differences should be taken into account when designing studies which involve pair-feeding to control nutrient intake.

Acute Disease↗
PubMed1997

Alterations in oxidative metabolism and glutamine transport support glucose production in the tumor-influenced hepatocyte.

Glutamine is the primary substrate whose hepatic transport is upregulated in the tumor-bearing host; however, the subsequent metabolism of transported glutamine is currently unknown. The purpose of this study was to determine if glutamine is an important oxidative fuel source for hepatocytes in cancer. Specifically we compare rates of glutamine transport and oxidation in hepatocytes from control and tumor-bearing animals. We also compare rates of glucose oxidation and rates of glucose production from glutamine in control hepatocytes versus those from tumor-bearing animals. Hepatocytes from rats bearing the MCA fibrosarcoma were isolated when tumors comprised 5 and 15% of total body weight and compared to sham-implanted and pair-fed control animals. [3H]GLN transport, GLN and glucose oxidation to CO2, and glucose production from glutamine were assayed. Tumor burden of 5% stimulated a 2.52-fold increase in hepatocyte glutamine transport and a 2-fold increase when tumor burden reached 15%. Rates of oxidation of glutamine were suppressed by 1.5-fold when tumors comprised 5% of body weight compared to sham animals and were equivalent to sham animals when tumors comprised 15% of body weight. Significant alterations in glucose oxidation were observed when tumors were both small and large-glucose oxidation was suppressed by 3.6- and 3.7-fold when tumors comprised 5 and 15% of body weight respectively compared to sham-implanted rats. Incubation of hepatocytes from tumor-bearing animals with glutamine as a gluconeogenic substrate induced a 1.84-fold increase in glucose production compared to sham hepatocytes. In conclusion, (i) despite a doubling of GLN transport by the tumor-influenced hepatocyte, GLN oxidation by hepatocytes was not increased. (ii) Glucose oxidation by hepatocytes from tumor-bearing animals was decreased compared to sham hepatocytes and, simultaneously, glucose production by tumor-influenced hepatocytes from glutamine was increased. The augmentation of hepatic glutamine transport and decreased glutamine oxidation seen in tumor-influenced hepatocytes appear to support hepatocyte gluconeogenesis from glutamine.

Animals↗
PubMed1997

Characterization of glutamine and glutamate transport in rat lung plasma membrane vesicles.

Insufficient glutamine for the lungs during sepsis may contribute to an impairment in lung function. Lung glutamine metabolism is supported by both blood glutamine uptake and de novo biosynthesis using circulating glutamate as a precursor. Information regarding the specific plasma membrane carriers involved in this uptake is lacking. Furthermore, the effect of sepsis on amino acid transport in whole lung has not been studied. We isolated lung plasma membrane vesicles (LPMVs) from control and LPS-treated rats and assayed glutamine and glutamate transport activity in LPMVs. Vesicle purity and functionality were confirmed by time-dependent concentrative amino acid uptake in the presence of Na+, impoverishment of microsomal enzymes, and a 25-fold enrichment in the plasma membrane marker 5'-nucleotidase. Eighty percent of glutamine uptake in lung vesicles was mediated via the high affinity Na(+)-dependent carrier System ASC (Vmax = 80 +/- 10 pmole/mg protein/15 sec; Km = 224 +/- 30 microM) while 19% occurred via the Na(+)-independent System ASC (Vmax = 11 +/- 2 pmole/mg/15 sec; Km = 141 +/- 23 microM). Ninety percent of glutamate transport was mediated by the Na(+)-independent System XAG-. Treatment of rats with LPS resulted in a decrease in both glutamine and glutamate transport in LPMVs. LPMVs offer a novel method for characterizing lung amino acid transport and studying the effects of catabolic states on this activity. The effects of endotoxin on System ASC and XAG- activity may contribute to reduced lung glutamine availability during septic states which may impair cellular metabolism and function.

5'-Nucleotidase↗
PubMed1997

The multidisciplinary structured clinical instruction module as a vehicle for cancer education.

BACKGROUND: The Structured Clinical Instruction Module (SCIM) modifies the Objective Structured Clinical Examination (OSCE) for teaching purposes. This study determined the effectiveness of a breast cancer SCIM in enhancing residents' clinical skills. METHODS: Twenty-five residents, 15 faculty members, and 12 breast cancer patients (simulated and actual) participated in the multistation, multidisciplinary SCIM. Afterward, faculty members, residents, and patients evaluated the SCIM. Residents completed an 18-item self-assessment of their skills before and after the SCIM. RESULTS: All residents, faculty members, and patients rated the SCIM as either outstanding or above average as an educational experience. The residents' self-assessments of their skills were significantly higher after the SCIM than before. CONCLUSIONS: This study shows that residents are aware of their deficiencies in breast cancer management. The SCIM provides an excellent format for residents to improve their clinical skills.

Attitude of Health Personnel↗
PubMed1997

Improving residents' clinical skills with the structured clinical instruction module for breast cancer: results of a multiinstitutional study. Breast Cancer Education Working Group.

BACKGROUND: The purpose of this study was to determine, in a multiinstitutional setting, the effectiveness of the structured clinical instruction module (SCIM) as an instructional format for surgical residents. METHODS: The breast cancer SCIM is an abbreviated (3-hour) clinical skills course that places residents in realistic clinical settings. The curriculum encompasses all aspects of breast cancer patient assessment. The SCIM was administered to 137 residents at five institutions. Sixty-six faculty members and 52 patients participated. All participants were surveyed with multiitem questionnaires. The residents were also asked to perform a self-assessment of their skills before and after the SCIM. RESULTS: The SCIM was delivered at all institutions without difficulty. All participants rated the SCIM highly (from "above average" to "outstanding"). Mean ratings (on a 5-point scale) for the overall effectiveness of the SCIM as an educational format follow: [table: see text] The pretest mean (on a 5-point scale) on the self-assessment was 2.46 ("less than competent"); the posttest mean was 3.54 ("more than competent") (p < 0.0001). CONCLUSIONS: Residents are acutely aware of their deficiencies in understanding breast cancer. The SCIM is a standardized, reproducible, portable, and effective educational vehicle.

Breast Neoplasms↗
PubMed1997

Regulation of glutamine synthetase in human breast carcinoma cells and experimental tumors.

BACKGROUND: Acute deprivation of extracellular glutamine causes up-regulation of glutamine synthetase (GS) expression by a mechanism involving an increase in GS protein stability. This study examines GS expression in a highly glutamine-dependent and tumorigenic human breast cancer cell line, TSE cells, in response to acute and chronic glutamine deprivation in culture and during tumor formation. METHODS: TSE cells were subjected to acute glutamine deprivation, adapted to growth in low glutamine concentrations, and subcutaneously injected into nude mice. GS protein and mRNA levels were assayed by Western and Northern blotting, and intracellular glutamine levels were evaluated by using a colorimetric assay. RESULTS: GS protein levels increased, but GS mRNA levels were unchanged in response to acute glutamine deprivation. Chronic glutamine deprivation in vitro and tumor growth in vivo caused an increase in both GS protein and mRNA levels. Large tumors exhibited lower intracellular glutamine, higher GS protein, and relatively unchanged GS mRNA levels relative to small tumors. CONCLUSIONS: TSE tumors exhibit up-regulation of GS protein and mRNA levels and declines in intracellular glutamine content, suggesting that growth in vivo causes a chronic and progressive glutamine deprivation. Up-regulation of GS expression may contribute to adaptation to a nutrient-poor intratumor environment.

Animals↗
PubMed1997

Surgical management of primary cutaneous melanomas of the hands and feet.

OBJECTIVE: The purpose of the study was to investigate the surgical management of cutaneous melanomas of the hands and feet. SUMMARY BACKGROUND DATA: Prior studies suggest that patients with melanomes > 1-mm thick should be treated with excision with a 2-cm margin and undergo elective lymphadenectomy in selected circumstances. These recommendations are based primarily on data from melanomas of the trunk and extremities. Melanomas of the hands and feet are less common and less well studied. They pose a surgical challenge because primary wound closure often is difficult, and the incidence and management of regional node metastases are unclear. METHODS: Charts of patients with melanomas of the hands or feet treated at the Massachusetts General Hospital between 1980 and 1994 were reviewed retrospectively. Local recurrence rates and the incidence of regional node metastases were analyzed as a function of histology, margin of excision, and microscopic thickness of the melanoma. RESULTS: Data from 116 patients (39 men, 77 women) with melanomas of the hands (n = 26) and feet (n = 90) were evaluated. Pathologic diagnoses were: acral lentiginous melanoma (48 patients); subungual melanoma (13 patients), and skin of dorsum of the hand or foot (n = 55). Digital amputation was required in all 13 patients with subungual melanoma to maintain local control; still, nodal metastases developed in 46% of patients within 1 year. Seventy-one percent of patients with acral lentiginous melanoma presented with lesions > or = 1.5 mm, and nodes or systemic disease or both developed in 56% of patients. Acral lentiginous melanoma lesions < 1.5-mm thick were treated principally by excision with a 1-cm margin; a local recurrence or metastases did not develop in any of the patients. None of the patients with melanomas on the dorsum of the hand or foot < 1.5-mm thick had a local recurrence, but regional or systemic disease developed in > 50%. Local control in patients with lesions > 1.5-mm thick frequently required skin grafting or amputation. The majority of patients with melanomas > or = 1.5 mm in thickness undergoing elective lymph node dissection had histologically positive nodes for melanoma. CONCLUSIONS: Melanomas of the hands and feet < 1.5-mm thick have a low incidence of nodal metastases and are treated effectively with wide excision of the primary with a 1-cm margin. Thicker melanomas are associated with a > 50% rate of regional or systemic failure. In the absence of metastatic disease, these individuals should undergo local excision with a 2-cm margin and intraoperative lymphatic mapping followed by lymphadenectomy if the sentinel node is positive.

Adolescent↗
PubMed1997

Does information from axillary dissection change treatment in clinically node-negative patients with breast cancer? An algorithm for assessment of impact of axillary dissection.

OBJECTIVE: The authors assessed the impact of axillary dissection on adjuvant systemic therapy recommendations in patients with breast cancer. SUMMARY BACKGROUND DATA: With increasing use of systemic therapy in node-negative women and the desire to reduce treatment morbidity and cost, the need for axillary dissection in clinically node-negative patients with breast cancer has been challenged. METHODS: Two hundred eighty-two women with clinically negative axillae were analyzed using a model treatment algorithm. Systemic therapy was assigned with and without data from axillary dissection. Treatment shifts based on axillary dissection data were scored. RESULTS: Twenty-seven percent of clinically node-negative women had pathologically positive nodes. Eight percent of T1a and 10% of T1b tumors had positive nodes and would have been undertreated without axillary dissection. Seven percent of premenopausal women with tumors < 1 cm and 13% with tumors > or = 1 cm had treatment changed by axillary dissection. For women 50 to 60 years of age, 10% with tumors < 1 cm, 17% with tumors 1 to 2 cm with positive prognostic features, and 4% with poor prognostic features had significant treatment shifts after axillary dissection. For clinically node-negative women older than 60 years of age not eligible for chemotherapy, only 3% of those with tumors < 1 cm and none of those with tumors > or = 1 cm had their treatment changed by findings at axillary dissection. Treatment shifts based on axillary dissection were larger if the treatment algorithm allowed for more varied or more aggressive treatment options. CONCLUSIONS: Data obtained from axillary dissection will alter adjuvant systemic therapy regimen in a significant number of clinically node-negative women younger than 60 years of age and for older women eligible to receive chemotherapy.

Adult↗