Peter Thomas

The effect of transfection of the CEA gene on the metastatic behavior of the human colorectal cancer cell line MIP-101

Carcinoembryonic antigen (CEA) has been shown to increase the metastatic potential of some human colorectal cancer cell lines. To investigate further the mechanisms involved we have produced three clones (6, 8 and 17) from the poorly differentiated human colorectal cancer cell line MIP-101 that have been transfected with the full length cDNA encoding for human carcinoembryonic antigen (CEA). They produce CEA with a mol. wt. of 180000 by Western blotting and secrete it into the culture medium. Clone 6 is a high CEA producer, clones 8 and 17 are intermediate producers. The doubling time for clone 8 was similar to that of the parent cell line while clones 6 and 17 had doubling times nearly twice that of the parent cells. These clones are tumorigenic when injected subcutaneously in nude mice are positive for CEA by immunoperoxidase staining and the mice have elevated blood levels of CEA. Clone 6 formed large aggregates in culture while clone 17 formed smaller aggregates. Clone 8 behaved like the parent cell line with rare cell/cell contact. Clones 6 and 17 also adhered to CEA coated plastic while clone 8, a neo-transfected control and the parent cell line did not. A significant increase in the incidence of hepatic tumors was observed with clone 6 (P < 0.01) and clone 17 (P < 0.02) following intrasplenic injection into nude mice. Immunohistopathology of the hepatic tumors showed strong CEA staining from clones 6 and 17 with weak staining from clone 8. The parent cell line was negative for CEA as were the neo-transfected controls. Of the neo controls none of 10 had liver colonies. Mice injected with clone 6 which developed liver metastasis had the highest plasma levels of CEA (37.3 +/- 8.8 ng/ml). We observed strong CEA staining in Kupffer cells in the normal liver adjacent to the CEA producing tumors. This study provides further evidence for the involvement of CEA in the metastatic process.

Role of the liver in clearance and excretion of circulating carcinoembryonic antigen (CEA)

CEA is a glycoprotein with a molecular weight of 200,000 containing 55%–65% carbohydrate. The removal of only two sialic acid residues result in rapid uptake from the circulation by the liver and catabolism in the lysosomes. There is a receptor on the plasma membrane of the hepatocyte (hepatic binding protein) which recognizes galactosyl residues. About 70% of125I-labeled intact CEA is cleared by the liver in 1 hr. The exposure of terminal galactose residues by removing sialic acids determines the rate of clearance. CEA is probably initially taken up by Kupffer cells and transferred to hepatocytes. About 10% of CEA added to an isolated perfused liver appears in bile. Biliary duct obstruction and cholestasis may elevate plasma CEA levels due to detergent effects on the liver cell receptors.

Clinical Correlations of α2,6-Sialyltransferase Expression in Colorectal Cancer Patients

We have previously demonstrated a link between α2,6-Sialyltransferase (α2,6-ST; E.C. 2.4.99.1) expression and differentiation of colon tumors. So far, information is not available relative to the e...

Hepatic mechanisms for clearance and detoxification of bacterial endotoxins.

This review attempts to cover the present state of knowledge of the physiologic mechanism present in the liver for the clearance and detoxification of endotoxins. The liver as a clearance site. Endotoxin-macrophage interactions and hepatic detoxification of endotoxin; Kupffer cells (KC), other macrophages and hepatocytes. KC: hepatocyte communication. Gene expression and regulation by bacterial endotoxins.

Modification of antibody isoelectric point affects biodistribution of 111-indium-labeled antibody.

We evaluated the influence of changes in charge on the biodistribution of 111In-labeled purified rabbit antihuman serum albumin (R-HSA) IgG conjugated to diethylenetriaminepentaacetic dianhydride (DTPA). Optimization of isoelectric point (pI) may influence the biodistribution profile, especially retention in vital organs, which ultimately affects radioimmunoimaging. Experiments were designed to modify the pI of R-HSA by conjugating various molar ratios of DTPA (DTPA:R-HSA ratios 5:1 to 100:1). The pI of the conjugates was determined by isoelectricfocusing (IEF). 111In-labeled DTPA:R-HSA with known pI range was injected intraperitoneally into BALB/c mice to evaluate biodistribution. There was a proportional relationship between the molar ratio of DTPA to R-HSA IgG and the number of DTPA substituted. Molar ratios of 5, 10, 20, 50 and 100 gave, on average, 2.0, 3.6, 5.1, 9.5 and 16.0 DTPA per R-HSA IgG, respectively. An anodal shift in the pI of 111In-labeled R-HSA IgG was noted with increased number of DTPA conjugation. Biodistribution studies at both 4 and 24 h showed sequential increase in the liver activity with increasing number of DTPA per antibody, whereas colon and small intestine showed a decrease in the activity at 4 h. The organ-specific increase (e.g., liver) or decrease (e.g., colon and small intestine) in the activity may depend on a critical balance of charge of a particular organ and its interaction with the amount of negative charge carried by the antibody conjugate. The results suggest that pI optimized 111In-labeled antibody could be used to increase or decrease colon and hepatic retention for more efficient radioimmunoimaging of colon tumors and their hepatic metastasis.

Characterization of a new monoclonal antibody to a cell surface antigen on colorectal cancer and fetal gut tissues

Murine hybridoma were raised against the human colon carcinoma cell line CL‐187. One clone was found to secrete a monoclonal antibody (ND‐1) that recognizes a large external antigen (LEA) on human colon carcinoma cells. With indirect immunofluorescence on formaldehyde‐fixed cells, more than 90% of the human colorectal carcinoma cell lines tested expressed LEA. Almost all of the 46 human noncolorectal and nonhuman cell lines tested did not express LEA, including cancer cell lines from other endodermally derived tissues. Staining of frozen sections from human colorectal tumors, noncolorectal tumors, normal adult, and normal fetal tissues showed expression of the antigen on colorectal cancer tissue, fetal colon, and fetal biliary epithelium. LEA can also be detected in the serum and ascites of colorectal cancer patients. Double indirect immunofluorescence with rabbit anti‐carcinoembryonic antigen (CEA) antibody and ND‐1 monoclonal antibody on a human colorectal carcinoma cell line showed that LEA is distinct from CEA. Physicochemical analysis of LEA showed that it has a large molecular weight, is resistant to extraction from the cell surface, and that sialic acid is an important component of the antigenic site. Because of the specificity for colorectal cancer tissue along with certain biochemical properties, LEA appears to be unique when compared with other tumor‐associated antigens. Further research is needed to define the clinical usefulness of LEA in either the diagnosis or treatment of colorectal carcinoma.

Serum β-N-acetyl hexosaminidase (β-NAH) as a discriminant between malignant and benign extrahepatic biliary obstruction: Comparison with carcinoembryonic antigen (CEA)

Fifty-one patients (16 with malignant extrahepatic biliary obstruction, ten with benign extrahepatic biliary obstruction, eight with alcoholic liver disease, five with viral hepatitis and 12 with liver metastases) and 19 adult healthy controls were studied with determinations of beta-N-acetyl hexosaminidase (a lysosomal enzyme which is cleared from the circulation by the Kupffer cells), carcinoembryonic antigen (CEA), serum bilirubin, alkaline-phosphatase and aspartate aminotransferase (AST). Both CEA and beta-NAH were elevated in each disease group. Elevated beta-NAH levels distinguished between benign and malignant extrahepatic biliary obstruction better than CEA levels. Beta-NAH levels for the malignant and the benign groups were 47.6 +/- 14.7 U/l and 23.0 +/- 4.7 U/l (mean +/- S.D.) respectively. The groups differed significantly (P less than 0.001). Plasma CEA levels for both groups were 18.7 +/- 38.9 and 7.2 +/- 3.3 ng/ml (mean +/- S.D.) respectively. Beta-NAH levels for the 19 normal controls were 15.8 +/- 3.5 U/l (mean +/- S.D.). Beta-NAH also was significantly elevated in patients with hepatic metastases (36.9 +/- 20.1 U/l). In 25 cancer patients with metastases other than in the liver beta-NAH levels (18.3 +/- 5.2) were not significantly elevated over the control group. It has potential value as a marker for non-CEA-producing liver metastases.

Selective Tumor Uptake of Boronophenylalanine-Fructose (BPA-F) in an Animal Model of Hepatic Colorectal Metastases

In 1998 more than 150,000 Americans will be diagnosed with colorectal cancer, and over 60,000 will die from this tumor. At the time of diagnosis, 15–25% will have hepatic metastases, and another 20–30% will develop metastatic hepatic lesions subsequent to resection of the primary tumors. Treatment options for hepatic metastases include aggressive surgical resection, administration of regional chemotherapeutic agents, sys temic chemotherapy, radiotherapy, or other palliative and experimental procedures. Despite well-defined pharmacological benefits of selected chemotherapeutics, there is no convincing survival advantage for many of the above mentioned techniques.1,2,3,4 The median survival after diagnosis is typically 22–24 months, and has changed minimally over the past decade.