Carcinoembryonic antigen: a rat model.

It has been shown that a TAA termed rat CEA had the tissue distribution and physico-chemical properties similar to those of human CEA. In addition, it has been demonstrated that these two antigens shared antigenic determinants. These findings supported our contention that rat CEA and human CEA are analogous moieties. The production of CEA-specific autoantibodies and the induction of resistance to CEA-positive rat tumors after immunization of rats with extracts containing rat CEA raises the possibility that human CEA may be immunogenic in man. Treatment with heat, proteolytic enzymes and periodate oxidation revealed that rat CEA, similar to human CEA contained both carbohydrate and protein epitopes. The epitopes shared by rat and human CEA that were detectable by the monkey anti-human CEA serum appeared to be carbohydrate, whereas the epitopes on rat CEA with which the rat mAb combined appeared to be protein, and those detected by the rabbit anti-rat CEA serum appeared to be carbohydrate, as well as protein. These studies also indicated that, although the rat, rabbit and monkey produced antibodies specific for rat CEA, the epitopes detectable by antibodies from one species appeared to be distinct from those detectable by antibodies from the other two species. These observations have important implications in studies on human CEA. Its use as a reliable diagnostic marker for malignancy hinges on the detection of tumor-specific epitopes on the molecule. Such epitopes have not yet been clearly identified by antibodies produced in foreign species. Indeed, our finding that the rat mAb to rat CEA bound only to tumor extracts and not to extracts of normal tissues, including intestinal tissues, suggests that human beings would be the most likely source of a tumor-specific antibody to human CEA. In future studies, the role antibodies play in immunity against CEA-positive tumors will be explored and attempts will be made to determine whether all of the serologically detectable epitopes on rat CEA can induce tumor resistance, or whether this activity is limited to epitopes detectable only by rat antibodies. This information could have important implications in the use of human CEA in the immunotherapy of malignancies.

Serological relationship between rat and human carcinoembryonic antigen.

Enzyme immunoassays showed that a rabbit antiserum to rat carcinoembryonic antigen (CEA) had high activity with extracts containing human CEA, which was almost completely inhibited by extracts containing human or rat CEA. Little or no inhibition was obtained with extracts of normal human or rat tissues or by human blood group A or B substances. A monkey antiserum to human CEA had strong activity against extracts containing rat CEA, and little or no activity against extracts of normal rat liver or kidney or a CEA-negative rat colon tumor. Activity of the monkey antiserum against rat CEA was almost completely inhibited by extracts containing rat or human CEA. Little or no inhibition was obtained with extracts of normal rat or human tissues. These results suggest that human and rat CEA share antigenic determinants and that they may be analogous moieties.

The effect of sodium butyrate and retinoic acid on growth and CEA production in a series of human colorectal tumor cell lines representing different states of differentiation.

The effect of sodium butyrate and retinoic acid added singly or in combination on substrate-dependent growth, colonization efficiency in soft agar, and carcino-embryonic antigen (CEA) production in three human colorectal carcinoma cell lines differing in their degree of differentiation was studied. All three colon cancer cell lines regardless of their state of differentiation had their growth markedly slowed by sodium butyrate, and to a lesser extent by retinoic acid. When both agents were added together, a small synergistic inhibition of growth was noted in all the cell lines. Butyrate eliminated colony formation in soft agar in all three cell lines, however, retinoic acid only reduced colony formation in the well differentiated cell line DLD-2. Sodium butyrate was able to induce CEA production in the undifferentiated cell (MIP-101) and the moderately differentiated cells (clone D) which were previously negative for this marker. It also enhanced the baseline production of CEA in the well differentiated cells (DLD-2). Retinoic acid did not induce CEA production in clone D or MIP-101 cells, but did enhance the production of CEA in DLD-2 cells. When both retinoic acid and sodium butyrate were added together, CEA production was either additive (DLD-2) or was inhibited (clone D and MIP-101). One explanation of these results is that only well differentiated cells have functional cellular retinoic acid-binding protein (cRABP), and that certain actions of retinoic acid (inhibition of anchorage-dependent growth) are independent of the presence of cRABP.

Isolation and characterization of an undifferentiated human colon carcinoma cell line (MIP-101).

An undifferentiated human colon carcinoma cell line was established from tumor tissue obtained from metastasis to the liver of colonic adenocarcinoma in a patient with fulminant Dukes D colorectal carcinoma. Histological analysis of the tumor biopsy from the liver confirmed the hospital pathology report of poorly differentiated colonic adenocarcinoma. Explants of this tumor tissue xenografted into a nude mouse were used to establish an epithelioid-like cell culture line, MIP-101. The cell line formed tumors in nude mice that histologically appeared undifferentiated and did not stain for carcinoembryonic antigen (CEA). No CEA was present either by radioimmunoassay (RIA) of the culture supernatant or by immunoperoxidase staining of the tumors or monolayers. MIP-101 appears to be one of the most undifferentiated human colon carcinoma cells lines available. It should prove useful in the search for markers of undifferentiated colonic cancer and in studies of colonic cancer differentiation.

Regulation of cellular immune response against autologous human melanoma. I. Evidence for cell-mediated suppression of in vitro cytotoxic immune response.

The potential existence of down-regulation of cytotoxic immune response against an autologous human melanoma line was investigated as a possible explanation for cytotoxic unresponsiveness against the autologous melanoma cells. The melanoma cell line, PJ-M, was established and lymph node resident lymphocytes (LNL) were isolated from a lymph node which was partially infiltrated with the melanoma cells. Autologous peripheral blood lymphocytes (PBL) were sensitized in in vitro co-culture (IVC) against radiated PJ-M cells in the presence or absence of PJ-M-sensitized LNL and enriched suppressor (OKT8+) or inducer (OKT4+) LNL populations, and were assayed for cytotoxicity in a 4-hr 51Cr-release microcytotoxicity assay. Significant cytotoxic response against PJ-M could be generated in the PBL, but not in the LNL. The addition of sensitized, unfractionated LNL, OKT8+, or OKT4+ LNL populations abrogated cytotoxic response in the PBL against PJ-M. The suppression of cytotoxic response was induced selectively against the PJ-M targets, because IVC of PBL in the presence of the sensitized LNL did not affect the generation of polyclonal cytotoxic alloreactivities, nor did they abrogate the generation of cytotoxic response against allogeneic targets in IVC against the corresponding allogeneic targets. These results suggest the possibility that cytotoxic immune response against the autologous melanoma cells might have been suppressed by the individual's own immunoregulatory circuit.

Tumor immunity in rats immunized with rat carcinoembryonic antigen.

Active immunization of rats with an emulsion consisting of Freund's complete adjuvant (FCA) and an extract of rat tumor containing carcinoembryonic antigen (CEA) induced clear-cut protection from growth of the syngeneic CEA-positive tumor, RCA-1. No protection was observed in rats treated with FCA alone nor was there protection against a tumor that no serologically detectable CEA. The results suggested that the tumor immunity exhibited by the immunized rats was mediated by an immune response specific for rat CEA. It was shown further that multiparous rats were more resistant to growth of RCA-1 tumor than nulliparous rats. This suggested that immunization against rat CEA, which is an oncofetal antigen, may occur during pregnancy.

In vitro cytotoxicity and transplantation protection by autologous natural and activated killer cells against an in vitro transformed tumorigenic fibroblast line. A case study.

Cytotoxic immune response by autologous natural killer (NK) cells against a spontaneous in vitro transformed tumorigenic fibroblast line, VIP-F:T, was studied in a 4 h 51Cr-release microcytotoxicity assay and in a tumor cell neutralization technique in vivo in nude mice. Although highly cytotoxic against the NK prototype target K562, the autologous NK cells in their nascent state were only marginally cytotoxic against VIP-F:T and unreactive against the autologous normal fibroblasts, Pen-F2. Autologous NK activity against VIP-F:T could, however, be induced by 2-16-h treatment of the NK cells with several species of interferon and by interferon-free interleukin 2 (IL-2). In vitro co-culture (IVC) in IL-2 of autologous peripheral blood lymphocytes (PBL) against VIP-F:T was shown by fluorescence activated cell sorting and by cold target competition experiments to generate almost exclusively an effector population bearing HNK-1 and Leu-11a phenotypes which exhibited receptor specificity for VIP-F:T distinct from receptors on Pen-F2 or K562 cells. PBL, co-cultured in IL-2 against Pen-F2 or K562, or cultured in IL-2 alone, generated high levels of nonspecific killing and showed no receptor specificity. Identical IVC in IL-2 of autologous PBL against a melanoma line, VIP (PBL and the VIP line derived from the same patient from whom the VIP-F:T line was also derived), and similar IVC in IL-2 of several other autologous PBL against their corresponding target cell lines (established from surgical specimens) generated cytotoxic responses involving cytotoxic populations bearing T8 as well as HNK-1 phenotypes; but the cytotoxic activities in none of these systems showed target receptor specificity. Autologous PBL, co-cultured against VIP-F:T in IL-2, were shown to be capable of rejecting tumorigenic challenge with VIP-F:T.3 (a clone of VIP-F:T) in nude mice at effector to VIP-F:T ratio of 10:1. The protective effect of the co-culture activated PBL was abrogated if the HNK-1+ cells were depleted from the effector population. Our data, thus, demonstrate specificity of cytotoxic reactivity which, by phenotypic markers, can be characterized as HNK-1 and Leu 11a+ cells under these experimental conditions against this particular in vitro transformed VIP-F:T line. In addition, this study shows that similar studies of cytotoxic autologous reactivities against in vitro transformed target cell lines will provide valuable information on the subject of NK-mediated surveillance against human neoplasia.

Induction of antibodies in rats to a rat carcinoembryonic antigen.

In a previous communication, carcinoembryonic antigen (CEA) of rat, which is an analogue to human CEA, was demonstrated in gastrointestinal adenocarcinomas induced in inbred Fischer rats by injection of 1,2-dimethylhydrazine. Antibodies detectable by enzyme immunoassay were elicited in Fischer rats by immunization with a perchloric acid extract of the CEA-containing rat tumor, RCA-1, incorporated into Freund's complete adjuvant. Specificity studies showed that activity of the rat antisera could be virtually abolished by inhibition with the tumor extract used at a concentration of 25 micrograms/ml. Inhibition by newborn rat tissues required extract at a concentration of 250 micrograms/ml. Extracts of normal adult tissues did not inhibit at these concentrations, but did inhibit at a concentration of 2,500 micrograms/ml. The results showed that rat CEA, though present in low concentration in normal adult rat tissue, is capable of eliciting an immune response in rats.