Generation and characterization of anti-MUC4 monoclonal antibodies reactive with normal and cancer cells in humans.

We have previously cloned the full-length cDNA (approximately 28 Kb) and established the complete genomic organization (25 exons/introns over 100 kb) of the human MUC4 mucin. This large molecule is predicted to protrude over 2 microm above the cell surface, in which MUC4alpha is an extracellular mucin-type glycoprotein subunit and MUC4beta is the transmembrane subunit. Over two thirds of the encoded protein sequence consists of 16-amino-acid tandem repeats (TR), which are flanked by unique sequences. In this study we generated and characterized monoclonal antibodies (MAbs) directed against the TR region of MUC4. Mice were immunized with a KLH-conjugated MUC4 TR peptide, STGDTTPLPVTDTSSV. Several clones were purified by three rounds of limited dilutions and stable clones presenting a sustained antibody production were selected for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the MUC4 peptide and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the MAbs (8G7) was strongly reactive against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis. Anti-MUC4 MAb may represent a powerful tool for the study of MUC4 function under normal and pathological conditions and for diagnosis of solid tumors including those in the breast, pancreas, lungs, and ovaries.     

Identification and sequencing of the Syrian Golden hamster (Mesocricetus auratus) p16(INK4a) and p15(INK4b) cDNAs and their homozygous gene deletion in cheek pouch and pancreatic tumor cells.

Previous studies have shown that the p16(INK4a) tumor suppressor gene is inactivated in up to 98% of human pancreatic cancer specimens and 83% of oral squamous cell carcinomas. Inactivation of the related p15(INK4b) gene has also been identified in a number of tumors and cell lines, however, its role as an independent tumor suppressor remains to be elucidated. Chemically-induced tumors in the Syrian Golden hamster (Mesocricetus auratus) have been shown to be excellent representative models for the comparative development and progression of a number of human malignancies. The purpose of this study was to determine the importance of the p16(INK4a) and p15(INK4b) genes in two experimental hamster models for human pancreatic and oral carcinogenesis. First, hamster p16(INK4a) and p15(INK4b) cDNAs were cloned and sequenced. The hamster p16(INK4a) cDNA open reading frame (ORF) shares 78%, 80%, and 81% identity with the human, mouse, and rat p16(INK4a) sequences, respectively. Similarly, the hamster p15(INK4b) cDNA ORF shares 82% and 89% sequence identity with human and mouse p15(INK4b), respectively. Second, a deletion analysis of hamster p16(INK4a) and p15(INK4b) genes was performed for several tumorigenic and non-tumorigenic hamster cell lines and revealed that both p16(INK4a) and p15(INK4b) were homozygously deleted in a cheek pouch carcinoma cell line (HCPC) and two pancreatic adenocarcinoma cell lines (KL5B, H2T), but not in tissue matched, non-tumorigenic cheek pouch (POT2) or pancreatic (KL5N) cell lines. These data strongly suggest that homozygous deletion of the p16(INK4a) and p15(INK4b) genes plays a prominent role in hamster pancreatic and oral tumorigenesis, as has been well established in correlative studies in comparable human tumors. Furthermore, this study supports the comparative importance of the hamster pancreatic and cheek pouch models of carcinogenesis in subsequent mechanistic-, therapeutic-, and preventive-based studies aimed at providing important translational data applicable to pancreatic adenocarcinoma and oral squamous cell carcinoma in humans.     

ANTIPYRETIC ACTION OF DEXAMETHASONE ON EGTAZIC ACIDINDUCED FEVER IN RABBITS

本文用脑室灌注和Ｆｕｒａ２测定细胞内游离钙技术观察了地塞米松（ｄｅｘａｍｅｔｈａｓｏｎｅ,ＤＥＸ）对家兔乙二醇双（２氨基乙醚）四乙酸性发热效应和下丘脑细胞内游离钙浓度（［Ｃａ２＋］ｉ）的影响,借此深入探讨地塞米松解热作用的中枢机制。结果发现：脑室灌注乙二醇双（２氨基乙醚）四乙酸（０６ｎｍｏｌ）引起家兔结肠温度明显升高,静脉注射地塞米松（５ｍｇ／ｋｇ）显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,地塞米松（６０～１２０μｍｏｌ／Ｌ）并不影响下丘脑细胞内［Ｃａ２＋］ｉ,而事先脑室灌注抑制基因转录的放线菌素Ｄ（３ｎｍｏｌ）则完全取消了地塞米松对乙二醇双（２氨基乙醚）四乙酸性发热的解热作用。这些结果提示：地塞米松显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,其机制与地塞米松激活脑内某些基因的表达有关,而与下丘脑神经细胞跨膜钙离子流无关。     

Biphenyl sulfonic acid ligands for catalytic C-N cross coupling of aryl halides with anilines and secondary amines

The use of two biphenyl sulfonic acid ligands for the catalytic C-N cross coupling of aryl halides with anilines, 3-aminopyridine, and secondary amines is reported. Our results represent a significant improvement compared to state of the art methods especially with regards to the removal of palladium.     

Engineering and characterization of a divalent single-chain Fv angiotensin II fusion construct of the monoclonal antibody CC49

For the therapy of solid tumors, co-administration of angiotensin II (AngII) results in an increased uptake of drugs into the tumor interstitium. We have engineered a dimeric sc(Fv)(2)-AngII fusion construct that combines the superior kinetics of covalent dimeric scFvs [sc(Fv)(2)], recognizing the pancarcinoma tumor-associated antigen 72 (TAG-72), with the advantageous intrinsic activity of AngII. The binding characteristics of the fusion construct were unaltered by the addition of the AngII sequence [affinity constant K(A) 1.18 x 10(7) and 8.42 x 10(6) M(-1) for sc(Fv)(2) and sc(Fv)(2)-AngII, respectively]. The binding of the fusion construct to the angiotensin receptor (AT(1)) was similar to AngII, and the arterial contraction was 16 +/- 1% of the response observed with norepinephrine. In animal studies, the radiolabeled sc(Fv)(2)-AngII construct exhibited similar uptake and a more homogeneous distribution within the tumor as compared to sc(Fv)(2).     

Expression of nerve growth factors in pancreatic neural tissue and pancreatic cancer.

One of the characteristics of pancreatic cancer is its tendency to invade neural tissue. We hypothesized that the affinity of cancer cells for nerve tissue is related to the presence of growth factors in neural tissue and their receptors in cancer cells. Sections of pancreatic cancer and normal pancreatic tissue were examined by immunohistochemistry for the expression of the neurotrophins NGF, BDNF, NT-3, NT-4, and their receptors TrkA, TrkB, and TrkC, as well as the low-affinity receptor, p75NTR. TrkA expression was found in duct, islet, and cancer cells; TrkB was found in the alpha-cells of the islet only. The anti-pan-Trk antibody (TrkB3), which is presumed to recognize all three receptors, immunoreacted with duct and acinar cells in normal tissue and with cancer cells. The staining with TrkC was similar to that of TrkA. The low-affinity receptor p75NTR was expressed in the neural tissue and in scattered duct cells of the normal tissue only. Duct and acinar cells, as well as neural tissue and cancer cells, showed weak to strong immunoreactivity with NGF. NT-3 expression was noted in capillary endothelia and erythrocytes. NT-4 showed specific staining for ductule cells. The expression and distribution of neurotrophins and their receptors suggest their role in the potential of pancreatic cancer cells for neural invasion.     

Pathobiological implications of MUC16 expression in pancreatic cancer

MUC16 (CA125) belongs to a family of high-molecular weight O-glycosylated proteins known as mucins. While MUC16 is well known as a biomarker in ovarian cancer, its expression pattern in pancreatic cancer (PC), the fourth leading cause of cancer related deaths in the United States, remains unknown. The aim of our study was to analyze the expression of MUC16 during the initiation, progression and metastasis of PC for possible implication in PC diagnosis, prognosis and therapy. In this study, a microarray containing tissues from healthy and PC patients was used to investigate the differential protein expression of MUC16 in PC. MUC16 mRNA levels were also measured by RT-PCR in the normal human pancreatic, pancreatitis, and PC tissues. To investigate its expression pattern during PC metastasis, tissue samples from the primary pancreatic tumor and metastases (from the same patient) in the lymph nodes, liver, lung and omentum from Stage IV PC patients were analyzed. To determine its association in the initiation of PC, tissues from PC patients containing pre-neoplastic lesions of varying grades were stained for MUC16. Finally, MUC16 expression was analyzed in 18 human PC cell lines. MUC16 is not expressed in the normal pancreatic ducts and is strongly upregulated in PC and detected in pancreatitis tissue. It is first detected in the high-grade pre-neoplastic lesions preceding invasive adenocarcinoma, suggesting that its upregulation is a late event during the initiation of this disease. MUC16 expression appears to be stronger in metastatic lesions when compared to the primary tumor, suggesting a role in PC metastasis. We have also identified PC cell lines that express MUC16, which can be used in future studies to elucidate its functional role in PC. Altogether, our results reveal that MUC16 expression is significantly increased in PC and could play a potential role in the progression of this disease.