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5-Fluorouracil (5-FU) and human recombinant gamma-interferon (gamma-IFN) were found to increase the expression of carcinoembryonic antigen (CEA) in human cancer cells in vitro. In the present study, the antimetabolite was associated with gamma-IFN or folinic acid (FA), a biochemical modulator of cellular metabolism of 5-FU, able to increase its antineoplastic activity. Treatment of two human colon cancer cell lines (HT-29 and WiDr) with 5-FU + gamma-IFN resulted in an increase of CEA expression higher than that obtainable with both agents alone, although no synergistic effects were obtained. This was demonstrated in terms of: (a) mRNA transcripts (HT-29); (b) cytoplasm and membrane CEA protein levels detected by Western blot analysis (HT-29); and (c) plasma membrane reactivity determined by flow cytometry analysis (HT-29 and WiDr). Moreover, 5-FU + gamma-IFN increased HLA class I molecules in the HT-29 cell membrane over that obtainable with gamma-IFN alone. In contrast, both agents did not induce the expression of the costimulatory molecule B7-1. Treatment with FA enhanced the antitumor effect of 5-FU but not its ability to augment CEA expression. This suggests that the FA-sensitive biochemical mechanism of action of 5-FU is not involved in its effect on CEA expression. In vivo studies showed, for the first time, that 5-FU, alone or combined with gamma-IFN, increases the amount of CEA protein over controls, either in cancer cells or in peripheral blood of nude mice bearing HT-29 cells. These results could be of potential diagnostic and/or therapeutic value when CEA protein is the target of humoral or cell-mediated immunity.

5-Fluorouracil (5-FU) and human recombinant γ-interferon (γ-IFN) were found to increase the expression of carcinoembryonic antigen (CEA) in human cancer cells in vitro. In the present study, the antimetabolite was associated with γ-IFN or folinic acid (FA), a biochemical modulator of cellular metabolism of 5.FU, able to increase its antineoplastic activity. Treatment of two human colon cancer cell lines (HT-29 and WiDr) with 5-FU + γ-IFN resulted in an increase of CEA expression higher than that obtainable with both agents alone, although no synergistic effects were obtained. This was demonstrated in terms of: (a) mRNA transcripts (HT-29); (b) cytoplasm and membrane CEA protein levels detected by Western blot analysis (HT-29); and (c) plasma membrane reactivity determined by flow cytometry analysis (HT-29 and WiDr). Moreover, 5-FU + γ-IFN increased HLA class I molecules in the HT- 29 cell membrane over that obtainable with γ-IFN alone. In contrast, both agents did not induce the expression of the costimulatory molecule B7-1. Treatment with FA enhanced the antitumor effect of 5-FU but not its ability to augment CEA expression. This suggests that the FA-sensitive biochemical mechanism of action of 5-FU is not involved in its effect on CEA expression. In vivo studies showed, for the first time, that 5-FU, alone or combined with γ-IFN, increases the amount of CEA protein over controls, either in cancer cells or in peripheral blood of nude mice bearing HT-29 cells. These results could be of potential diagnostic and/or therapeutic value when CEA protein is the target of humoral or cell-mediated immunity.

Fotemustine is a cytotoxic alkylating agent, belonging to the group of nitrosourea family. Its mechanism of action is similar to that of other nitrosoureas, characterized by a mono-functional/bi-functional alkylating activity. Worth of consideration is the finding that the presence of high levels of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT) in cancer cells confers drug resistance. In different clinical trials Fotemustine showed a remarkable antitumor activity as single agent, and in association with other antineoplastic compounds or treatment modalities. Moreover, its toxicity is generally considered acceptable. The drug has been employed in the treatment of metastatic melanoma, and, on the basis of its pharmacokinetic properties, in brain tumors, either primitive or metastatic. Moreover, Fotemustine shows pharmacodynamic properties similar to those of mono-functional alkylating compounds (e.g. DNA methylating drugs, such as Temozolomide), that have been recently considered for the management of acute refractory leukaemia. Therefore, it is reasonable to assume that this agent could be a good candidate to play a potential role in haematological malignancies.

Comparative studies on the suppressive effects of recombinant interferon-alpha (IFN-alpha), 5-fluorouracil (5-FU), or IFN-alpha + 5-FU have been performed in vitro on colon carcinoma cells (HT-29 cell line) and PHA-stimulated mononuclear cells (MNC) of peripheral blood obtained from healthy donors. IFN-alpha was used at 500 U/ml against HT-29 cells and at 1000 U/ml against MNC on day 1 of culture; 5-FU was used at 250 microM against HT-29 and at 1400 microM against MNC on day 2 of culture. The results show that: (a) IFN-alpha inhibited MNC and HT-29 cells by 13.4% and 32.9%, respectively; (b) 5-FU inhibited MNC and HT-29 cells by 54.7% and 87.0%, respectively; (c) IFN-alpha + 5-FU resulted in a stronger inhibition of HT-29 cells (i.e., 96.1%). In contrast, that combination was significantly less suppressive than 5-FU alone when MNC were used as targets (i.e., 35.9% inhibition). Natural cell-mediated cytotoxic activity relative to 10(6) MNC was not markedly altered by all agents alone or in combination. Moreover, treatment with IFN-alpha, 5-FU or IFN-alpha + 5-FU resulted in a marked increase in the number of HT-29 cells positive for the CEA surface antigen. These data seem to provide further rational support of the clinical use of IFN-alpha + 5-FU in colorectal cancer, based on the differential toxicity of this drug combination on tumor versus normal immunocompetent cells.

Residual telomerase activity (TA) could be used as a marker of malignant or normal cell survival after exposure to cytotoxic agents. Therefore TA after treatment with ionizing radiation was used in a radiosensitivity assay.Radiosensitive MOLT-4 and relatively radioresistant Jurkat leukemia cells or normal peripheral blood mononuclear cells (MNC) were irradiated with 5-160 Gy. Cell count, MTT assay and telomerase activity were evaluated on day 3 and clonogenic assay on day 10.In Jurkat cells, irradiation inhibited tumor growth and total TA per culture (TA/culture), but up-regulated TA per viable cell (TA/cell). TA/culture and TA/cell were profoundly depressed by irradiation of MOLT-4 or phytohemagglutinin-activated MNC.Susceptibility of normal or neoplastic cells to high-dose radiation can be monitored by evaluating residual TA/culture. In some cases, however, difficulties in the interpretation of the results could stem from radiation-induced increase of TA/cell, as observed for the Jurkat cell line.

Summary: Twelve patients with metastatic colorectal cancer received alternating cycles of low immunomodulat-ing doses of alpha-IFN + 5-Fluorouracil (5-FU) or 5-FU alone. Hematological, biochemical and physical evaluation showed that both treatment cycles were well tolerated. However, transient fever and moderate flu-like symptoms were observed following alpha-IFN administration. Treatment with 5-FU alone produced long-lasting inhibition of CD8+ T lymphocytes, but did not depress NK activity (NKA). Combined treatment with alpha-IFN produced a short-term increase of NKA and antagonized the effect of 5-FU on CD8+ cells on day 5 of the cycle. Parallel studies on in vitro models showed antiproliferative effects of 5-FU on PHA-stimulated MNC and confirmed the preferential inhibition of CD8+ cells. Pretreatment with alpha-IFN did not reverse the effect of 5-FU on CD8+ lymphocytes, but partially protected MNC from the toxic effects of the drug. This was presumably due to the cytostatic effects induced by alpha-IFN on MNC before exposure to the cycle-specific antineoplastic agent. This investigation suggests that alpha-IFN could play a positive role in immuno-chemotherapy of colorectal cancer through multiple mechanisms not entirely related to direct antitumor effects of the agent. © 1991 Kluwer Academic Publishers.

Treatment with 5-fluorouracil (5-FU) or recombinant interferon-gamma (IFN), alone or in combination, was found to increase carcinoembryonic antigen (CEA) expression in several carcinoma cell lines. In this study we examined the in vitro effect of these agents on CEA expression of tumor cells, obtained from a patient operated for rectal cancer. The results showed that exposure of cancer cells to 5-FU or to IFN resulted in increased CEA levels in terms of percentage of CEA-positive cells and mean fluorescence values, as indicated by FACS analysis. However, drug combination did not induce CEA expression higher than that provided by single agents alone. Treatment with 5-FU or with IFN produced a reduction of the total number of viable cells. Moreover, Western blot analysis revealed that exposure of cancer cells to each drug was followed by a substantial increase of the total cellular CEA content. On the contrary, 5-FU in combination with IFN did not increase the expression of the antigen more than that obtained by single agents. Noteworthy, exposure of CEA-negative cells from adjacent normal rectal tissue to both agents alone or in combination, did not result in CEA induction. In conclusion, the present results suggest new approaches aimed at (a) increasing the sensitivity of diagnostic procedures based on detection of CEA-positive tumor cells; (b) facilitating the recognition of CEA-positive cancer cells by immune responses induced by anti-CEA peptide vaccines.

The antitumor activity of hexamethylmelamine (HMM) was tested in various mouse tumor models in the presence or absence of host-vs-tumor graft responses. The drug was moderately active against Sarcoma-180 growing in different strains of non-sensitized mice. Strong protection was afforded when recipients were preimmunized with irradiated tumor cells 15 days before tumor challenge followed by HMM treatment. The drug did not show antitumor activity against two radiation-induced lymphomas of congenic mice of B10 background, inoculated into H-2 compatible hosts, or into mice incompatible for subregions of H-2. In this model HMM increased mortality of allogeneic mice presumably through impairment of host-vs-lymphoma graft resistance. In conclusion this study shows that synergistic or antagonistic effects can be obtained by combining chemotherapy with antitumor immune responses.

Previous studies showed that 5-fluorouracil (5-FU) is capable of enhancing the membrane reactivity of the human colon carcinoma cell line HT-29 with a monoclonal antibody (COL-1) directed against carcinoembryonic antigen (CEA). In the present study, we show that short-term exposure (i.e., 1 hr) of cancer cells to 5-FU mediates a marked increase of CEA expression, that is concentration-dependent and lasts up to day 5 after treatment. This phenomenon is the result of the drug-mediated enhancement of the CEA expression, but not of the selection of the CEA-positive cells operated by the antimetabolite. This is supported by the finding that the increase of the CEA expression detected by cytofluorimetric analysis is observed not only in the parental HT-29 line, but also in its C22.20 subclone, endowed with a low basal level of CEA and with chemosensitivity to 5-FU lower than that of the parental cell line. Moreover, increase of CEA expression occurs not only in the plasma membrane, but also in the cytosolic cellular compartment, as indicated by the results of Western blot analysis. Northern blot analysis of total RNA extracted from 5-FU-treated HT-29 or C22.20 cells shows an increase in the steady-state levels of CEA and CEA-related transcripts (e.g., biliary glycoprotein). Moreover 5-FU-mediated augmentation of the CEA transcript appears to be attributable mainly to enhanced transcription rather than to increased mRNA stability. It is concluded that induction of enhanced CEA protein expression in cancer cells treated with 5-FU could be of clinical interest for the development of immunochemotherapeutic protocols based on CEA protein as the target molecule.

(51)Cr-prelabelled colon cancer cells (simulating 'circulating tumor cells', CTCs) were added to human peripheral blood and exposed to staurosporine (ST) to increase carcinoembryonic antigen (CEA) expression. CTCs were captured with immunomagnetic beads coated with Ber-EP4 monoclonal antibody, recognizing the common epithelial antigen present in the majority of cancer cells of epithelial origin, with capture efficiency of more than 80%. Moreover, ST treatment increased CEA expression without compromising Ber-EP4 capture efficiency. In a pilot clinical study on 37 patients, CTCs were captured using Ber-EP4 beads, and recognized by RT-PCR set for CEA or cytokeratin-19 (CK) mRNA detection. The results showed that: (a) the percentage of CEA-positive CTCs (CTC(CEA), 54.1%) was lower than that of CK-positive CTCs (CTC(CK), 70.3%); (b) in vitro ST treatment converted a significant number of CTC(CEA)-negative into CTC(CEA)-positive cases. Therefore, immunomagnetic capture combined with exposure to ST provides a feasible and sensitive technique for the detection of functionally-active CTCs responsive to ST-mediated CEA up-regulation.

The availability of sensitive methods has allowed the detailed study of circulating tumor cells only recently. Evolving evidence support the prognostic and predictive role of these cells in patients affected by several solid tumors, including colorectal cancer. Ongoing studies are aimed at confirming that the molecular characterization of circulating tumor cells in peripheral blood and in bone marrow of patients is a powerful tool to improve the patient risk-stratification, to monitor activity of the drugs, to develop more appropriate targeted therapies and tailored treatments. In parallel, results from these correlative studies promise to gain a better biological understanding of the metastatic process. The clinical utility of the detection of circulating tumor cells in patients affected by colorectal cancer is still hampered by a number of specific hurdles. Improvement in sensitivity and specificity of the available methods of detection, standardization of these methods and functional characterization of circulating tumor cells in well designed and statistically well powered studies are the key steps to reach these ambitious objectives in colorectal cancer patients as well.

Summary Twelve patients with metastatic colorectal cancer received alternating cycles of low immunomodulating doses of alpha-IFN + 5-Fluorouracil (5-FU) or 5-FU alone. Hematological, biochemical and physical evaluation showed that both treatment cycles were well tolerated. However, transient fever and moderate flu-like symptoms were observed following alpha-IFN administration. Treatment with 5-FU alone produced long-lasting inhibition of CD8+ T lymphocytes, but did not depress NK activity (NKA). Combined treatment with alpha-IFN produced a short-term increase of NKA and antagonized the effect of 5-FU on CD8+ cells on day 5 of the cycle. Parallel studies on in vitro models showed antiproliferative effects of 5-FU on PHA-stimulated MNC and confirmed the preferential inhibition of CD8+ cells. Pretreatment with alpha-IFN did not reverse the effect of 5-FU on CD8+ lymphocytes, but partially protected MNC from the toxic effects of the drug. This was presumably due to the cytostatic effects induced by alpha-IFN on MNC before exposure to the cycle-specific antineoplastic agent. This investigation suggests that alpha-IFN could play a positive role in immuno-chemotherapy of colorectal cancer through multiple mechanisms not entirely related to direct antitumor effects of the agent.