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EB1089 completely inhibited growth of AT-84 SCC cells at nanomolar concentrations, reduced tumor growth, and did not have calcemic effects. Our results support continued investigation of EB1089 as a chemopreventive/chemotherapeutic agent for head and neck SCC.

We have analyzed interaction of coactivators with the wild-type estrogen receptor ␣ (ER), HEG0, and a mutant, L536P-HEG0, which is constitutively active in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. Different classes of coactivators do not recognize the ER ligand binding domain (LBD) in the same manner. Steroid receptor coactivator-1 (SRC-1), amplified in breast cancer-1 (AIB-1), transcriptional intermediary factor-1 (TIF-1), transcriptional intermediary factor-2 (TIF-2), and receptor interacting protein 140 (RIP140) interacted with HEG0 and L536P-HEG0 in the presence of estradiol, but generally not in the presence of anti-estrogens. However, ICI164,384 stimulated some interaction of RIP140 with LBDs. SRC-1, AIB-1, and RIP140 interacted constitutively with the L536P ER, whereas TIF-1 and TIF-2 interacted only weakly in the absence of hormone. Reciprocal competition for binding to the ER LBD was observed between different classes of coactivators. Moreover, coexpression of RIP140 blocked enhanced transactivation by HEG0 observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. We conclude that constitutive activity of L536P-HEG0 is manifested to similar degrees in different cell types and likely arises from constitutive coactivator binding; different classes of coactivators recognize distinct but overlapping binding sites on the ER LBD. Finally, the observation that L536P-HEG0 interacted constitutively with AIB-1, a coactivator that has been implicated in ER signaling in breast and ovarian cancer, suggests that similar mutations in the ER may contribute to hormone-independent proliferation of breast and ovarian cells. The estrogen receptor (ER)1 is a member of the family of nuclear receptors (1-6). Similar to other nuclear receptors, the ER is a ligand-activated regulator of transcription that functions through stimulating formation of transcriptional preinitiation complexes. Preinitiation complexes include general transcription factors, RNA polymerase II, and multiple components of polymerase II holoenzyme (7-10). The ER stimulates the assembly of these components through interaction with factors collectively known as coactivators that interact with the receptor ligand binding domain (LBD) in the presence of hormone (11). This interaction requires the AF-2 activating function, located at the extreme C terminus of the LBD.

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Analogs of 1alpha,25-dihydroxyvitamin D(3) (1alpha, 25(OH)2D3) inhibit growth in vitro and in vivo of cells derived from a variety of tumors. Here, we examined the effects of 1alpha,25(OH)2D3 and its analog EB1089 on proliferation and target gene regulation of human head and neck squamous cell carcinoma (SCC) lines SCC4, SCC9, SCC15, and SCC25. A range of sensitivities to 1alpha,25(OH)2D3 and EB1089 was observed, from complete G0/G1 arrest of SCC25 cells to only 50% inhibition of SCC9 cell growth. All lines expressed similar levels of vitamin D3 receptor (VDR) mRNA and protein, and no significant variation was observed in 1alpha,25(OH)2D3-dependent induction of the endogenous 24-hydroxylase gene, or of a transiently transfected 1alpha,25(OH)2D3-sensitive reporter gene. The antiproliferative effects of 1alpha,25(OH)2D3 and EB1089 in SCC25 cells were analyzed by screening more than 4,500 genes on two cDNA microarrays, yielding 38 up-regulated targets, including adhesion molecules, growth factors, kinases, and transcription factors. Genes encoding factors implicated in cell cycle regulation were induced, including the growth arrest and DNA damage gene, gadd45alpha, and the serum- and glucocorticoid-inducible kinase gene, sgk. Induction of GADD45alpha protein in EB1089-treated cells was confirmed by Western blotting. Moreover, while expression of proliferating cell nuclear antigen (PCNA) was reduced in EB1089-treated cells, coimmunoprecipitation studies revealed increased association between GADD45alpha and PCNA in treated cells, consistent with the capacity of GADD45alpha to stimulate DNA repair. While 1alpha,25(OH)2D3 and EB1089 modestly induced transcripts encoding the cyclin-dependent kinase inhibitor p21(waf1/cip1), no changes in protein levels were observed, indicating that p21(waf1/cip1) induction does not contribute to the antiproliferative effects of 1alpha,25(OH)2D3 and EB1089 in SCC cells. Finally, in partially resistant SCC9 cells, there was extensive loss of target gene regulation (10 of 10 genes tested), indicating that resistance arises from widespread loss of 1alpha,25(OH)2D3-dependent gene regulation in the presence of normal levels of functional VDRs.

Although widely used, DMSO is toxic for pancreatic islets. We combined hydroxyethyl starch (HES) with DMSO to simplify the procedure of freezing and thawing, and to decrease the toxicity of DMSO. A preclinical study was performed using islets from beagle dogs. After storage for 4 weeks, the islets were thawed and examined. The islet structure was well maintained after thawing. Although the number of the islets decreased to 71.2 +/- 20.1%, the function of the islets was evaluated by static incubation after thawing and showed a 1.80 +/- 0.78 stimulation index. We have introduced this technique for the cryopreservation of human islets from non-heart-beating donors. Twelve cases of human islet cryopreservation were performed. The sample tube of each human cryopreservation was thawed to evaluate the morphology, contamination, and endocrine function. Although fragmentation was observed in five samples (41.6%), the other seven (58.4%) showed a normal structure when evaluated by microscopic and electron microscopic study. The stimulation index (SI) of static incubation deteriorated from 3.37 +/- 3.02 to 1.34 +/- 0.28 after thawing. We divided the thawed islets into two groups: group 1 (n=8), SI > 1.2; group 2 (n=4), SI < 1.2. The group 1 islets showed a higher rate of normal structure (87%) than did group 2 (25%). Moreover, the SI before cryopreservation was 4.01 +/- 3.57 in group 1, which was higher than the SI of 2.11 +/- 0.72 in group 2. Based on the good results from the preclinical study using a large-animal model, this method was introduced for clinical application. Even from the pancreata of non-heart-beating donors, a successful islet cryopreservation was achieved. However, the isolated islets with poor function should not be cryopreserved for transplantation.

Background/purpose Living-donor pancreas transplants (LDPs) were introduced at Chiba-East National Hospital in 2004, and 12 LDPs have been performed at this institution to date. Based on the outcome of these 12 LDPs, the efficacy and safety of LDPs are herein discussed. Methods Twelve diabetic patients underwent LDPs; ten had simultaneous pancreas and kidney transplants from living donors, one had pancreas transplant after a kidney transplant from a living donor, and one had a pancreas transplant alone from a living donor. The donors were parents or brothers and the ABO blood types were incompatible in three LDPs. The procedures for the donor and recipient operations were performed according to the technique established by the University of Minnesota. Bladder drainage was used in 11 recipients and enteric drainage was used in one patient. Tacrolimus, basiliximab, mycophenolate mofetil, and prednisone were used for induction and immunosuppressive treatment. A splenectomy, double-filtered plasmapheresis, and plasma exchange were added in the ABO-incompatible LDPs.

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