Differential involvement of glutathione S‐transferase mu 1 and multidrug resistance protein 1 in melanoma acquired resistance to vinca alkaloids

On account of its extreme intrinsic resistance to apoptosis and of its strong ability to become chemoresistant after a primary response to drugs, malignant melanoma (MM) is still a therapeutic challenge. We previously showed that glutathione S‐transferase mu 1 (GSTM1) acts in synergy with multidrug resistance protein 1 (MRP1) to protect GSTM1‐transfected human CAL1 melanoma cells from toxic effects of vincristine (VCR). Herein, we investigated the role of these proteins in the acquired resistance of CAL1 cells to vinca alkaloids (VAs). Resistant lines were established by continuous exposure (>1 year) of parental CAL1‐wt cells to VCR, vindesine (VDS), or vinorelbine (VRB): CAL1R‐VCR, CAL1R‐VDS, CAL1R‐VRB, respectively. All resistant lines displayed more than 10‐fold increase in resistance to their selection VA, and specifically expressed GSTM1. Suggesting a direct interaction between this protein and VAs, each VA specifically decreased the GSTM1‐mediated glutathione conjugation activity in cell lysates. Curcumin (GSTM1 inhibitor), BSO (glutathione synthesis inhibitor), and MK571 (MRP1 inhibitor) considerably reversed the acquired resistance to VCR and VDS, but not to VRB. Microarray data analysis revealed similar gene expression patterns of CAL1R‐VCR and CAL1R‐VDS, and a distinct one for CAL1R‐VRB. These data suggest a differential involvement of GSTM1 and MRP1 in acquired resistance to VAs. A coordinated expression and activity of GSTM1 and MRP1 is required to protect CAL1 cells from VCR and VDS, while the simple expression of GSTM1 is sufficient, possibly by a direct drug/protein interaction, to confer resistance against VRB.     

Imaging of neuroendocrine tumors: accuracy of helical CT versus SRS

Background: We retrospectively compared the accuracy of somatostatin receptor scintigraphy (SRS) with that of helical computed tomography (CT) in the detection and localization of primary and metastatic neuroendocrine tumors.Methods: A medical record search identified 27 patients with known or clinically suspected neuroendocrine tumors who underwent helical CT and SRS within 3 months of one another at our institution. CT images were evaluated retrospectively by two blinded radiologists who used consensus reading. Images were evaluated for the presence or absence of primary tumor and hepatic and extrahepatic metastases. CT results were compared with the SRS report as interpreted by the nuclear medicine physicians. The results of the surgical, clinical follow-up, and pathologic findings were considered as the gold standard. Sensitivity, specificity, and accuracy were calculated for both imaging techniques. In addition, McNemar analysis was performed to determine statistically significant differences between CT and SRS.Results: Helical CT was more sensitive than SRS in the detection of extrahepatic metastases, and the difference between the two imaging modalities was statistically significant (p = 0.0312) as determined by the McNemar chi-square test. However, the difference between CT and SRS in detecting primary neuroendocrine tumors, hepatic metastasis, and combined hepatic and extrahepatic metastasis was not statistically significant (p = 0.625, 1.000, and 1.000, respectively).Conclusion: Helical CT and SRS have similar sensitivity, specificity, and accuracy in detecting primary neuroendocrine tumor and hepatic metastasis. However, helical CT appears to be more sensitive in detecting extrahepatic metastasis from primary neuroendocrine tumors.     

Liver regeneration after living adult right lobe transplantation

BACKGROUND: The purpose of this study was to describe liver regeneration in patients undergoing living-adult liver transplantation. METHODS: This prospective study included 10 donors and eight recipients who had a total of 65 computed tomographic (CT) scans. All patients had preoperative CT ( n = 18), and follow-up CT scans ( n = 47) were obtained for up to 14 months after transplantation. Liver and spleen volumes were measured by hand tracing each organ on the axial portal venous phase images. RESULTS: Both donors and recipients showed immediate increases in liver volume. However, liver regeneration was significantly faster and reached a higher peak in recipients than in donors. Splenic volume in donors demonstrated an initial increase followed by a decline, reaching the preoperative volume after 1 year. Splenic volume in recipients demonstrated immediate decline postoperatively. CONCLUSION: Restoration of liver volume occurred rapidly after transplantation, but followed different patterns in donors and recipients. Deviation from these patterns warrants further investigation.     

SPAK Differentially Mediates Vasopressin Effects on Sodium Cotransporters

Activation of the Na⁺-K⁺-2Cl⁻-cotransporter (NKCC2) and the Na⁺-Cl⁻-cotransporter (NCC) by vasopressin includes their phosphorylation at defined, conserved N-terminal threonine and serine residues, but the kinase pathways that mediate this action of vasopressin are not well understood. Two homologous Ste20-like kinases, SPS-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive kinase (OSR1), can phosphorylate the cotransporters directly. In this process, a full-length SPAK variant and OSR1 interact with a truncated SPAK variant, which has inhibitory effects. Here, we tested whether SPAK is an essential component of the vasopressin stimulatory pathway. We administered desmopressin, a V2 receptor–specific agonist, to wild-type mice, SPAK-deficient mice, and vasopressin-deficient rats. Desmopressin induced regulatory changes in SPAK variants, but not in OSR1 to the same degree, and activated NKCC2 and NCC. Furthermore, desmopressin modulated both the full-length and truncated SPAK variants to interact with and phosphorylate NKCC2, whereas only full-length SPAK promoted the activation of NCC. In summary, these results suggest that SPAK mediates the effect of vasopressin on sodium reabsorption along the distal nephron.The furosemide-sensitive Na⁺-K⁺-2Cl⁻-cotransporter (NKCC2) of the thick ascending limb (TAL) and the thiazide-sensitive Na⁺-Cl⁻-cotransporter (NCC) of the distal convoluted tubule (DCT) are key regulators of renal salt handling and therefore participate importantly in BP and extracellular fluid volume homeostasis.¹ Loss-of-function mutants in the SLC12A1/ A3 genes encoding NKCC2 and NCC cause salt-losing hypotension and hypokalemic alkalosis in Bartter’s and Gitelman’s syndromes,^2,3 whereas their overactivity may contribute to essential hypertension.^4,5 Recently, attention has been focused on the two closely related STE20-like kinases, SPS-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1), which can phosphorylate NKCC2 and NCC at their N-terminal conserved threonine or serine residues (T96, T101, and T114 of mouse NKCC2 and T53, T58, and S71 of mouse NCC) and thereby activate the transporters.^6–8 Despite the high homology between SPAK and OSR1 and their overlapping renal expression patterns, distinct roles along the nephron have been suggested based on data from SPAK-deficient and kidney-specific OSR1-deficient mice: deletion of SPAK primarily impairs the function of NCC, whereas deletion of OSR1 negatively affects NKCC2.^9–11 The complex functional properties of a WNK-SPAK/OSR1-N(K)CC interaction cascade are currently being defined.¹² Recently, arginine vasopressin (AVP), signaling via the V2 receptor (V2R), has been identified as an efficient activator of both cotransporters, affecting their luminal trafficking and phosphorylation.^13–18 Because plasma AVP levels may vary not only with the sleep-wake cycle or long-term physiologic challenges, but also with pulsatile changes over the short term, distinct, time-dependent responses may occur.¹⁹ SPAK and OSR1 are well placed to regulate distal NaCl reabsorption in response to AVP. Here we tested the role of SPAK in AVP-induced activation of NKCC2 and NCC, acutely and during long-term treatment. The results suggest that SPAK is an essential kinase that modulates distal nephron function in response to AVP stimulation.