Blocks to thyroid cancer cell apoptosis can be overcome by inhibition of the MAPK and PI3K/AKT pathways.

Current treatment for recurrent and aggressive/anaplastic thyroid cancers is ineffective. Novel targeted therapies aimed at the inhibition of the mutated oncoprotein BRAF(V600E) have shown promise in vivo and in vitro but do not result in cellular apoptosis. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a tumor-selective manner by activating the extrinsic apoptotic pathway. Here, we show that a TRAIL-R2 agonist antibody, lexatumumab, induces apoptosis effectively in some thyroid cancer cell lines (HTh-7, TPC-1 and BCPAP), while more aggressive anaplastic cell lines (8505c and SW1736) show resistance. Treatment of the most resistant cell line, 8505c, using lexatumumab in combination with the BRAF(V600E) inhibitor, PLX4720, and the PI3K inhibitor, LY294002, (triple-drug combination) sensitizes the cells by triggering both the extrinsic and intrinsic apoptotic pathways in vitro as well as 8505c orthotopic thyroid tumors in vivo. A decrease in anti-apoptotic proteins, pAkt, Bcl-xL, Mcl-1 and c-FLIP, coupled with an increase in the activator proteins, Bax and Bim, results in an increase in the Bax to Bcl-xL ratio that appears to be critical for sensitization and subsequent apoptosis of these resistant cells. Our results suggest that targeting the death receptor pathway in thyroid cancer can be a promising strategy for inducing apoptosis in thyroid cancer cells, although combination with other kinase inhibitors may be needed in some of the more aggressive tumors initially resistant to apoptosis.

Hyperparathyroidism after thyroid surgery and autotransplantation of histologically normal parathyroid glands.

BACKGROUND: Parathyroid autotransplantation is a well-established method to prevent hypoparathyroidism during parathyroid and thyroid operations. The reported success rate of parathyroid autotransplantation ranges from 75% to 100%. Recurrent hyperparathyroidism may develop after parathyroid autotransplantation, especially after the transplantation of hyperplastic or adenomatous parathyroid tissue. Hyperparathyroidism recurs most frequently after subtotal parathyroidectomy or total parathyroidectomy and autotransplantation, in patients with renal failure and secondary hyperparathyroidism, and in patients with familial primary hyperparathyroidism or MEN I or MEN II syndrome. We report three patients who experienced primary hyperparathyroidism after autotransplantation of normal parathyroid tissue during thyroid operations (two patients) or after a long period of hypoparathyroidism. STUDY DESIGN: We reviewed our records from 1983 to May 1998 and identified three patients in whom hyperparathyroidism developed after thyroid operations. RESULTS: One patient had a thyroidectomy with left modified radical neck dissection for papillary thyroid cancer, followed by radioiodine ablative therapy. Two patients had thyroid operations for benign thyroid disease. One of these patients had a history of radiation exposure for acne, and in the other one secondary hyperparathyroidism arose 6 years after a thyroidectomy for hyperthyroidism. CONCLUSIONS: Our study documents that hyperparathyroidism may develop after autotransplantation of histologically normal parathyroid tissue and after a period of hypoparathyroidism after thyroid operations. For this reason, it is important to mark the site of the parathyroid transplantation.

Inhibition of prostate cancer neovascularization and growth by urokinase-plasminogen activator receptor blockade.

Binding of the serine protease urokinase (u-PA) to its receptor on tumor cell surfaces facilitates proteolysis and tumor invasion. We undertook this study to determine whether the role of u-PA in prostate cancer induced angiogenesis and secondary tumor growth by developing a homologous, immunocompetent in vivo model in which the tumors cells secrete an inhibitor of the murine u-PA receptor. A mutant recombinant murine u-PA that retains receptor binding but not proteolytic activity was made by PCR mutagenesis. Mutant u-PA and a reporter gene pRK luciferase were transfected and stably expressed in the highly metastatic rat Dunning MAT-LyLu prostate cancer cell line. Several clones expressing mutant u-PA and luciferase were identified by Western blotting, plasminogen zymography, and reverse transcription-PCR. One of these clones, 5C4, was injected s.c. into Copenhagen rats. Compared to animals injected with clones expressing pRK luciferase alone, tumors in animals injected with 5C4 cells were significantly smaller. Moreover, there were fewer lung micrometastases in the 5C4 animals. Primary tumor angiogenesis was measured by microvessel quantification of tissue stained with antibodies against von Willebrand factor. Mean microvessel density in 5C4 tumors was 4.3-fold lower than that in animals with tumors derived from the control tumor cell line (P < 0.0001). Significant inhibition of tumor growth was also observed for two additional MAT-LyLu cell lines expressing mutant u-PA. These findings suggest that cell surface u-PA contributes to prostate cancer growth by enhancing angiogenesis.

Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways.

The switch from a quiescent tumor to an invasive tumor is accompanied by the acquisition of angiogenic properties. This phenotypic change likely requires a change in the balance of angiogenic stimulators and angiogenic inhibitors. The nature of the angiogenic switch is not known. Here, we show that introduction of activated H-ras into immortalized endothelial cells is capable of activating the angiogenic switch. Angiogenic switching is accompanied by up-regulation of vascular endothelial growth factor and matrix metalloproteinase (MMP) bioactivity and downregulation of tissue inhibitor of MMP. Furthermore, we show that inhibition of phosphatidylinositol-3-kinase leads to partial inhibition of tumor angiogenesis, thus demonstrating that activated H-ras activates tumor angiogenesis through two distinct pathways. Finally, we show evidence for two forms of tumor dormancy.

Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol.

2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite which disrupts microtubule function, has been shown to inhibit proliferating cells in vitro and suppress certain murine tumors in vivo. In vitro screening has determined that breast cancer cell lines are most sensitive to inhibition by 2-ME. Additionally, 2-ME has been shown to inhibit angiogenesis in vitro. We tested whether 2-ME suppresses cytokine-induced angiogenesis in vivo and inhibits growth of a human breast carcinoma in severe combined immunodeficient mice. A model of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)-induced corneal neovascularization in C57BL/6 mice was used to evaluate the antiangiogenic effects of 2-ME and other microtubule inhibitors such as Taxol, vincristine, and colchicine. 2-ME (150 mg/kg p.o., n = 20) inhibited bFGF and VEGF-induced neovascularization by 39% and 54%, respectively. Taxol (6 mg/kg i.p., n = 17) inhibited bFGF and VEGF-induced neovascularization by 45% and 37%, respectively. Vincristine (0.2 mg/kg i.p., n = 8) and colchicine (0.25 mg/kg i.p., n = 8) had no effect. Treatment with 2-ME (75 mg/kg p.o., n = 9) for 1 month suppressed the growth of a human breast carcinoma in mice by 60% without toxicity. Recognition of the antiangiogenic and antitumor properties of 2-ME and Taxol may be crucial in planning clinical applications to angiogenesis-dependent diseases.

Antiangiogenic therapy of transgenic mice impairs de novo tumor growth.

Angiogenesis is activated during multistage tumorigenesis prior to the emergence of solid tumors. Using a transgenic mouse model, we have tested the proposition that treatment with angiogenesis inhibitors can inhibit the progression of tumorigenesis after the switch to the angiogenic phenotype. In this model, islet cell carcinomas develop from multifocal, hyperproliferative nodules that show the histological hallmarks of human carcinoma in situ. Mice were treated with a combination of the angiogenesis inhibitor AGM-1470 (TNP-470), the antibiotic minocycline, and interferon alpha/beta. The treatment regimen markedly attenuated tumor growth but did not prevent tumor formation; tumor volume was reduced to 11% and capillary density to 40% of controls. The proliferation index of tumor cells in treated and control mice was similar, whereas the apoptotic index was doubled in treated tumors. This study shows that de novo tumor progression can be restricted solely by antiangiogenic therapy. The results suggest that angiogenesis inhibitors represent a valid component of anticancer strategies aimed at progression from discrete stages of tumorigenesis and demonstrate that transgenic mouse models can be used to evaluate efficacy of candidate antiangiogenic agents.

Tumor suppressor loci on mouse chromosomes 9 and 16 are lost at distinct stages of tumorigenesis in a transgenic model of islet cell carcinoma.

Techniques that detect loss of genetic heterozygosity (LOH) have helped elucidate genes involved in human cancers. Previously, a genome-wide search using simple sequence length polymorphisms to detect LOH in islet cell tumors arising in a transgenic mouse model of multistage tumorigenesis had revealed two candidate tumor suppressor genes, Loh1 and Loh2, on chromosomes 9 and 16, respectively. We now have analyzed the early stages of tumor development in this model (hyperplastic, early angiogenic, and angiogenic islets) for LOH involving regions of chromosomes 9 and 16. On chromosome 9, hyperplastic and early angiogenic islets reveal a low rate of loss (< 5%) indistinguishable from background; angiogenic islets showed a 9% rate, whereas the final tumor stage had an 18% rate. By contrast, LOH was observed much earlier on chromosome 16. Notably, the LOH rate in angiogenic islets was 29%, comparable to the rate seen in end-stage tumors (32%). The results show that the two loci are lost preferentially at different stages of tumorigenesis. The observation that a high LOH rate at Loh2 is seen in the angiogenic islet stage suggests that this locus may contain an angiogenesis suppressor; in contrast, the later appearance of Loh1 may contribute to the progression from the angiogenic stage to a solid tumor. Tumors containing chromosomes with partial LOH have allowed improved localization of Loh1 to a region of approximately 3.2 centiMorgans on chromosome 9, syntenic with human chromosomes 3q and 15q.

A strategy to discover circulating angiogenesis inhibitors generated by human tumors.

The phenomenon of inhibition of tumor growth by tumor mass has been studied in many experimental animal systems and has been observed in several clinical scenarios. Not until the recent discovery of angiostatin, a circulating angiogenesis inhibitor generated in the presence of a murine Lewis lung tumor, has a satisfactory mechanism been proposed to explain this phenomenon. Thus far, no other animal or human tumors are known to generate angiostatin. In this study, we utilized a mouse corneal neovascularization model to detect circulating inhibitors of angiogenesis generated by three human tumors grown in immunodeficient mice: (a) the PC-3 human prostate carcinoma; (b) the CCL188 human colon carcinoma; and (c) the UBC urinary bladder carcinoma. Mice bearing these three primary tumors demonstrated significant inhibition of angiogenesis in the cornea induced by a pellet containing basic fibroblast growth factor. Corneas of mice bearing s.c. prostate and colon carcinomas showed significant inhibition of vessel length, clock-hours of neovascularization, and vessel density. However, corneas of mice bearing s.c. bladder carcinomas demonstrated significant inhibition of vessel density only. Three colon carcinomas (clone A, CX-1, and MIP101), the MDA-MB-435S breast carcinoma, the MM-AN melanoma, and the JE-3 choriocarcinoma did not significantly inhibit corneal neovascularization.

Hepatobiliary complications of polyarteritis nodosa.

Although polyarteritis nodosa (PAN) may result in thrombosis or aneurysm formation in any organ in the body, hepatobiliary complications are unusual. We reviewed seven cases that demonstrated the diagnostic difficulties and therapeutic options available in the management of hepatobiliary PAN. No consistent sign that indicated the severity of hepatobiliary PAN could be identified. In cases of thrombotic PAN, acalculus cholecystitis usually could be diagnosed preoperatively. Early tissue diagnosis and aggressive intervention are required for appropriate patient treatment. If the diagnosis is unclear, a preoperative muscle or skin biopsy specimen is often helpful in establishing a tissue diagnosis of PAN, even if no obvious pathologic condition is evident. Patients who undergo celiotomy for acalculus cholecystitis or peritoneal signs of an unclear origin should have tissue specimens (gallbladder wall, liver, or omentum) submitted for pathologic study. Angiography may be diagnostic preoperatively or when results of biopsies are equivocal. In addition, early angiography can define the extent of visceral involvement and permit control by embolization of hemorrhage secondary to aneurysm rupture. Awareness of the possibilities of thrombotic, ischemic, or bleeding complications from PAN allows more aggressive and rapid management of abdominal complaints, especially in patients who are receiving immunosuppressant therapy.

Tissue soldering by use of indocyanine green dye-enhanced fibrinogen with the near infrared diode laser.

Anastomoses welded by laser have been strengthened by applying a solder of fibrinogen combined with a laser energy absorbing dye (indocyanine green, maximum absorbance 805 nm) to the anastomotic site before continuous-wave diode laser exposure (808 +/- 1 nm, 4.8 W/cm2). Immediately after creation, the bursting pressures of welds created without fibrinogen (262 +/- 29 mm Hg, n = 11) were significantly less than repairs with fibrinogen (330 +/- 75 mm Hg, n = 11) (p less than 0.05). When repairs performed with fibrinogen were exposed to urokinase (25,000 IU) the bursting pressures were not significantly different from baseline (290 +/- 74 mm Hg, n = 5). Aortotomies closed by suture did not burst but leaked at pressures significantly below those of vessels closed by laser (165 +/- 9 mm Hg, n = 11) (p less than 0.01). Twenty-two repairs soldered with fibrinogen were incorporated into survival studies in rabbits and examined from 1 to 90 days after operation. No anastomotic ruptures, thromboses, or aneurysms were identified. Soldered sites rapidly regenerated a new intimal surface and healed by myofibroblast proliferation. No significant foreign body response was identified; the fibrinogen was resorbed. Laser soldering with exogenous fibrinogen is feasible without topical administration of additional clotting agents, significantly improves the bursting strength of primary laser welded anastomoses, and appears to result from urokinase-resistant fibrinogen cross-linking.

Strength of laser vascular fusion: preliminary observations on the role of thrombus.

We hypothesized that autologous clot deposited on the luminal surface of laser vascular welds immediately after creation would produce higher time zero bursting pressures that could be achieved in welds perfused with saline alone. To test this hypothesis, we compared bursting pressures of welds created in isolated rabbit aortic segments 1) with saline perfusion only, 2) with blood perfusion, and 3) with blood perfusion followed by infusion of urokinase. Tissue welds with saline perfusion had a mean bursting pressure of 159 +/- 45 mm Hg; tissue welds following blood perfusion had a mean pressure of 262 +/- 29 mm Hg; tissue welds with blood perfusion followed by urokinase infusion had a mean bursting pressure of 187 +/- 35 mm Hg. The saline and urokinase groups were not significantly different. However, the blood-perfused group was significantly higher than both the saline group and the urokinase group. Thus, the addition of urokinase eliminates the beneficial effects noted after blood reperfusion. These observations suggest that the enhancement of weld strength following exposure to blood is due predominantly to the adherence of fibrin-platelet aggregates at the site of the weld.

Role of calcium in mediating action of carbachol in T84 cells.

To examine the role of calcium in mediating carbachol's action in secretory epithelia, we simultaneously measured intracellular free [Ca] [( Ca]i) and transepithelial chloride transport in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value [( Ca]'i) normalized to control. Chloride transport was measured as the short-circuit current (Isc) with a voltage clamp. Monolayers were pretreated with cyclic AMP to augment the response of Isc to carbachol, a procedure that did not qualitatively change the response of the monolayer to carbachol. The carbachol-induced changes in Isc appeared to be dependent on the increase in [Ca]i. First, carbachol caused both Isc and [Ca]'i to increase in parallel. Isc increased from 32 +/- 5 to 70 +/- 9 microA and then declined to 57 +/- 16 microA while [Ca]'i increased from 72 +/- 14 to 156 +/- 22 nM and then declined to 133 +/- 45 nM. Second, the carbachol-induced increases in Isc and [Ca]'i were correlated. The greater the hormone-stimulated rise in [Ca]'i, the higher the increase in Isc. Third, carbachol's stimulation of Isc was blunted by preventing the calcium spike with the cellular calcium buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Although the carbachol-induced increase in [Ca]'i appeared necessary for the increase in Isc, it was not clear if carbachol's action was solely the result of an increase in [Ca]'i. Increasing [Ca]'i with ionomycin, although causing Isc and [Ca]'i to increase in parallel, failed to increase Isc to the levels observed with carbachol. These experiments suggest that although the carbachol-induced increase in Isc is dependent on the increase in [Ca]i, the hormone may activate a second process that increases the sensitivity of the calcium-activated transport process to changes in [Ca]i.

Variant surface glycoprotein gene expression site switches in Trypanosoma brucei.

In Trypanosoma brucei bloodstream forms, transcription of variant surface glycoprotein (VSG) genes occurs at only one of several possible expression sites at any given time. Activation and inactivation of some expression sites are correlated with recombinational events that alter their chromosomal position (Van der Ploeg, L. H. T., Cornelissen, A. W. C. A., Michels, P. A. M., and Borst, P. (1984) Cell 39, 213-221). We present evidence that a 430-kilobase pair (kb) chromosome, containing the 1.8 expression-linked copy, is taken up in a reciprocal recombination when the 1.8 gene is inactivated. As a result, the 430-kb chromosome is reduced in length either to 140 kb (in variant 118b') or to 350 kb (in variant MITat 1.2000) and the 1.8 expression-linked copy is moved to a larger chromosome in both cases. The subsequent activation of the telomeric 118 VSG gene in variant 118b', located on a 2000-kb chromosome, occurs without detectable recombinations while, for variant MITat 1.2000, still another expression site is activated. We discuss a model that explains the occurrence of these apparently random recombinational events at expression site switching and antigenic variation.