Preclinical studies on detection of circulating melanoma cells in patients: telomerase as a recognition marker of malignancy.

Preclinical studies based on a "simulation design", were performed with cultured melanoma cells prelabeled with 51Cr, added to normal blood and subjected to separation and recognition steps. Mononuclear cells (MNC) were isolated on ficollhypaque gradient, and melanoma cells were separated from lymphocytes using anti-CD45 immunomagnetic beads. Malignant cells were then recognized by measuring telomerase activity (TRAP and TRAP-ELISA assays). It was found that: (a)recovery of prelabeled cells present in MNC did not exceed 75%; (b) further recovery of prelabeled cells after separation from lymphocytes did not exceed 68%. Therefore, the overall recovery of prelabeled cells did not exceed 48%; (c) the entire procedure was able to reliably detect as few as 30 malignant cells added to normal blood, providing a telomerase signal significantly higher than that found in absence of melanoma cells. These results furnish the technical bases for developing a tumor detection assay in the blood of melanoma patients.

Adjuvant therapy of melanoma patients (stage II, III): a pilot immuno-toxicological study.

A pilot study was conducted to assess the tolerability and the effect on host immunity of a post-surgery adjuvant treatment of melanoma patients with an anti-angiogenic agent, Tamoxifen (TAM, 20 mg/die p.o., daily), combined with immunomodulating cytokines, i.e. recombinant interleukin-2 (IL-2, 4 MUI/m2 s.c., day 8,10,12) and alpha-2b-interferon (IFN, 3 MUI/m2 i.m., day 15,17,19), starting a new cycle on day 21, for a total of 12 cycles. Fifty patients (pts) entered into the study, 27 males and 23 females with a median age of 55 years (range 25-75), performance status (ECOG) 0 with melanoma stage IIA (12 patients), stage IIB (28 patients), stage III (10 patients). Preliminary in vitro studies showed that TAM does not interfere with up-regulation of natural immunity induced by IFN, IL-2, or IFN + IL-2 in normal peripheral blood mononuclear cells (MNC). The clinical study indicates that the protocol was well tolerated. Increase of NK and LAK activity of patient MNC was observed on day 15. The mean disease-free interval was 10 months and 40 pts were alive at 5 years of follow-up. Further investigations should be performed to test effectiveness of this protocol in a randomized study.

DNA repair enzymes and cytotoxic effects of temozolomide: comparative studies between tumor cells and normal cells of the immune system.

O6-alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair system (MRS) play a crucial role in the susceptibility of tumor cells to the cytotoxic effects of agents that generate O6-methylguanine in DNA, including the triazene compound temozolomide (TMZ). Studies performed with peripheral blood mononuclear cells (MNC) showed that TMZ was scarcely active on lymphocyte functions not dependent on cell proliferation (e.g. NK activity and cytokine-mediated induction of CD1b molecule in adherent MNC). In contrast, TMZ depressed proliferation and lymphokine activated killer (LAK) cell generation in response to IL-2. In this case, a reasonably good inverse relationship was found between OGAT levels of MNC and their susceptibility to TMZ. This study also analyzed the ratio of the toxic effect of TMZ on MNC and on tumor cells (i.e. "Tumor-Immune Function Toxicity Index", TIFTI). A particularly favorable TIFTI can be obtained when OGAT levels are extremely high in MNC and markedly low in tumor cells. This holds true for MRS-proficient neoplastic cells, but not for MRS-deficient tumors. In conclusion, strategies aimed at modulating OGAT and MRS may improve the clinical response to TMZ. However, the use of OGAT inhibitors to potentiate the antitumor activity of TMZ might result in a concomitant increase of the immunosuppressive effects of the drug, thus reducing the relative TIFTI.

A novel method for monitoring response to chemotherapy based on the detection of circulating cancer cells: a case report.

We describe a novel method for detecting micrometastasis in the blood stream of cancer patients based on RT-PCR amplification of tumor-associated carcinoembryonic antigen (CEA) mRNA. To increase sensitivity and specificity of RT-PCR, CEA transcript was selectively up-regulated in cancer cells by exposure of peripheral blood to non-toxic concentrations of staurosporine (ST). Thereafter, polyA(+) RNA was extracted from tumor cells captured by means of magnetic beads coated with a monoclonal antibody against a common human epithelial antigen. Finally, RNA was subjected to RT-PCR analysis of CEA transcript. Using this approach, we demonstrated an ST-mediated increase in CEA transcript in blood specimens collected from a patient with metastatic colon cancer before receiving treatment with 5-fluorouracil/leucovorin. After a few cycles of chemotherapy, CEA-positive tumor cells were no longer detected. Clinical follow-up of this patient indicated that treatment with chemotherapy induced a dramatic reduction in liver metastasis. Therefore, it can be hypothesized that lack of CEA transcript detection might be consistent with disappearance or at least marked reduction of circulating tumor cells.

Influence of Mycobacterium bovis bacillus Calmette Guérin on in vitro induction of CD1 molecules in human adherent mononuclear cells.

Nonpeptide antigens (including glycolipids of microbial origin) can be presented to T cells by CD1 molecules expressed on monocyte-derived dendritic cells. These HLA unrestricted responses appear to play a role in host immunity against Mycobacterium tuberculosis and other pathogenic bacteria. It is known that vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) has limited efficacy in many clinical settings, although the reasons for its inadequacy remain unclear. Here we have investigated the influence of BCG on the induction of CD1b on human monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF), which is believed to be the principal inducer of this antigen-presenting molecule. Although BCG alone led to a slight induction of CD1b expression, this agent reduced markedly the ability of GM-CSF to induce high levels of CD1b that were typically observed in uninfected cells. Inhibition of CD1b expression in BCG-infected monocytes was apparent at both the mRNA transcript and CD1b protein levels. Down-regulation of CD1b expression by BCG was mediated, at least in part, by one or more soluble factors and could not be reversed with high concentrations of GM-CSF or a variety of other cytokines. The present results suggest that BCG could diminish the efficiency of CD1-restricted T-cell responses against nonpeptide mycobacterial antigens by reducing CD1 expression on antigen-presenting cells. These findings have potential implications for understanding the nature of the immune response elicited by BCG in humans and suggest potential strategies that could be important for the development of better vaccines for the prevention of tuberculosis.

Bacillus Calmette-Guerin down-regulates CD1b induction by granulocyte-macrophage colony stimulating factor in human peripheral blood monocytes.

Non-peptide antigens (e.g. glycolipids of microbial origin) presented by monocyte-associated CD1 molecules to T cells appear to play an important role in host immunity against tuberculosis and other pathogenic bacteria. Since vaccination with Bacillus Calmette-Guerin (BCG) has limited efficacy, the influence of viable BCG organisms on the induction of CD1b antigen by granulocyte macrophage-colony stimulating factor (GM-CSF) has been tested in adherent mononuclear cells obtained from peripheral blood of healthy donors. The results indicate that the vaccine reduces substantially CD1b induction by GM-CSF. On the other hand, BCG was found to promote a slight increase in the expression of this molecule on target cells not exposed to GM-CSF. Attempts to reverse the antagonistic effects of BCG on GM-CSF with high concentrations of GM-CSF, alone, or associated with IL-4, were unsuccessful. Moreover, mycobacteria suppression by 10 microg/ml of rifampin, did not affect BCG influence on CD1b induction. The present results suggest that mycobacterium-induced impairment of the CD1 system could play a role in the unsatisfactory results obtained with BCG vaccination.

In vitro generation of cytotoxic T lymphocytes against HLA-A2.1-restricted peptides derived from human thymidylate synthase.

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.

Effect of 5-fluorouracil on carcinoembryonic antigen expression and shedding at clonal level in colon cancer cells.

BACKGROUND: The carcinoembryonic antigen (CEA) is a tumor marker largely utilized for the detection of minimal disease or as a target of immunotherapeutic approaches. In preclinical models CEA has been found to be up-regulated after exposure of cancer cells to 5-fluorouracil (5-FU). In the present study, the clonal distribution of CEA and its regulation by 5-FU at clonal level was investigated using human HT-29 colon cancer cells. MATERIALS AND METHODS: The extent of CEA expression was measured in terms of: (a) antigen levels on plasma membrane, by flow cytometry; (b) cytoplasm and membrane protein, by Western blot analysis: (c) transcript, by Northern blot analysis; (d) CEA shedding by radioimmunossay. RESULTS: CEA protein and gene transcript were variably expressed among different clones. In all cases 5-FU was able to increase the percentage of CEA-positive cells, the amount of antigen, either in the membrane or cytosolic fractions, and the corresponding transcript. Moreover, a marked increase of CEA shedding was found in drug-treated cells with respect to that of controls. The increase of CEA induced by the antimetabolite was not the result of a selection mechanism based on preferential killing of CEA negative cells. The antimetabolite was capable of enhancing antigen expression also in other CEA-positive tumor cell lines with different basal levels of the marker. CONCLUSIONS: The present findings could be of potential value to increase the sensitivity of diagnostic procedures based on detection of CEA positive tumor cells. Moreover, the antimetabolite might be included in immunotherapeutic protocols to facilitate recognition of CEA-positive cancer cells by immune responses.

In vitro effect of hyperthermia on natural cell-mediated cytotoxicity.

It is well known that hyperthermia (HY), which is used for the treatment of cancer, depresses natural cell-mediated immunity in vitro. Experiments were performed to confirm the inhibitory effect of HY (42 degrees C for 1 hour) on natural killer (NK) activity and to evaluate the influence of HY on the generation and cytotoxic activity of interleukin-2 (IL-2)-activated NK cells. Additional experiments were also carried out to evaluate the effect of a simultaneous exposure of effector and target cells to HY. The results showed that HY profoundly reduced the lytic activity of NK cells and demonstrated that this inhibition was transient and not due to an apoptosis-induced reduction of the number of effector cells. Moreover, the exposure of mononuclear cells to HY before IL-2 stimulation did not affect the generation of IL-2-activated NK cells, whereas, the hyperthermic treatment of IL-2-activated NK cells produced a marked reduction of their cytotoxic activity. The results also showed that the simultaneous exposure of effector and target cells to HY, during the cytotoxicity assay, produced a marked reduction of lytic activity of NK and IL-2-activated NK cells, and that this impairment was specific for effector cells. In this context, heat-exposure of target cells alone, did not substantially modify their susceptibility to lysis induced by either NK or IL-2-activated NK cells. These results add further evidence of HY-induced inhibition of natural cell-mediated immunity, and suggest that, in the course of therapeutic HY, immune response could be significantly altered.

Staurosporine increases carcinoembryonic antigen expression in a human colon cancer cell line.

Staurosporine (ST), a protein kinase C inhibitor, was found to produce antitumor effects against C22.20, a clonal subline derived from colon cancer HT-29 line, selected for low expression of carcinoembryonic antigen (CEA). However, as assessed by FACS analysis using propidium iodide, no apoptosis or cell cycle alteration was found on day 3 after treatment of C22.20 cells with ST (1-100nM). Exposure of cells to graded concentrations of the drug (i.e., from 1 to 25nM) resulted in a concentration-dependent increase in the percentage of CEA positive cells, as determined by flow cytometric analysis. However, when higher concentrations (i.e. 50nM - 100nM) of ST were used, the percentage of CEA positive cells declined compared to that detected in 25nM-treated tumor. Since these results were obtained in a clonal cell population, it is reasonable to hypothesize that induction rather than selection mechanism is involved in this phenomenon. The potential clinical interest of the present findings stems from the consideration that treatment with ST or its derivatives could improve sensitivity and efficacy of diagnostic and/or immunotherapeutic approaches based on CEA molecules.

Drug-induced modulation of carcinoembryonic antigen (CEA) expression in neoplastic cells from a patient with rectal cancer.

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.

Adjuvant treatment of breast cancer: a pilot immunochemotherapy study with CMF, interleukin-2 and interferon alpha.

Immune responses, including natural immunity (NI), potentiate the antitumor effects of chemotherapy. Since interferons and interleukin-2 (IL-2) augment NI, a pilot study was conducted to assess the tolerability and the effects on host immunity of adjuvant chemotherapy associated with IL-2 + interferon alpha (IFN) in breast cancer patients after surgery. Ten patients underwent alternating 28-day cycles of chemoimmunotherapy [cyclophosphamide + methotrexate + 5-fluorouracil (CMF, days 1, 8) + IL-2 (days 15 19) + IFN (day 22)] and chemotherapy alone (CMF). With this regimen each patient was considered to be a reasonable "control" of herself. Blood cell count and natural killer cell activity (NKA) were tested on days 1, 8, 15, 22, and 23. Preliminary in vitro studies indicated that IL-2 or IFN antagonized the severe inhibition of NKA induced by hydroxy-peroxy-cyclophosphamide (in vitro active derivative of cyclophosphamide), alone or associated with methotrexate + 5-fluorouracil. Nine patients completed all six alternating cycles, whereas one patient proved to have metastatic lesions after four cycles. The protocol was well tolerated, although leukopenia (CMF alone) and leukopenia with fever and moderate or minimal flu-like symptoms (CMF + IL-2 + IFN) were generally observed. Treatment with IL-2 facilitated complete recovery of white cell counts and NKA after the nadir on day 15. In conclusion, the present protocol appears to be well tolerated and amenable to administration on an outpatient basis. Therefore, further investigations should be performed to verify whether CMF + IL-2 + IFN would be superior to CMF alone for adjuvant treatment after surgery in breast cancer.

Effect of the combined treatment with 5-fluorouracil, gamma-interferon or folinic acid on carcinoembryonic antigen expression in colon cancer cells.

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.

Effect of rifampin on CD1b expression and double-negative T cell responses against mycobacteria-derived glycolipid antigen.

Non-classical antigen-presentation by CD1 molecules expressed on cytokine-activated monocytes (CAM), and cell-mediated responses supported by double-negative (DN) and by CD8+ responder alphabeta T cells, are involved in host resistance against mycobacterial infections. The CD1b protein is responsible for presentation of non-peptide, lipid antigens to T cells. In this context, a pivotal role is played by induction of CD1b protein on the membrane of human monocytes activated by GM-CSF alone, and more efficiently by GM-CSF combined with IL-4. Rifampin (RFP), a drug which is extensively utilized for chemoprophylaxis or treatment of Mycobacterium tuberculosis, is known to reduce a number of B, or T cell-dependent responses. Therefore we undertook immunopharmacological studies on RFP, to determine the effects of this agent on human macrophage function, relative to antigen presentation by CD1b molecules and on DN T cell cytolytic function. The results showed that: (a) graded concentration of RFP (2 or 10 microg/ml) induced a significant increase of CD1b expression, in CAM as evaluated by FACS analysis; (b) RFP increased significantly the specific mAb binding to CD1b on CAM surface; (c) treatment of effector cells with RFP did not reduce DN T cell-mediated cytolysis against lymphoblastoid cells transfected with CD1b cDNA (C1R.b6 cells), pulsed with M. tuberculosis. These results suggest that RFP could be of potential value in improving mycobacterial antigen presentation without impairing responder T cell function.

In vitro effects of growth hormone (GH) and insulin-like growth factor I and II (IGF-I and -II) on chromosome fragility and p53 protein expression in human lymphocytes.

BACKGROUND: We have reported previously that growth hormone (GH) therapy increases cell radiosensitivity; in this study we tested whether GH itself or IGFs induce chromosome aberrations and investigated the expression of p53 protein in response to DNA damage. METHODS: Human peripheral blood lymphocytes were incubated with GH [100 and 1000 microg L(-1)], insulin-like growth factor I [IGF-I; 150 and 1000 microg L(-1)] and IGF-II [600 and 1200 microg L(-1)] for 24 h. The radiomimetic agent bleomycin [BLM; 5 microgm L(-1)] was added in the last 3 h. Cytogenetic analysis was performed by assessing the percentages of damaged cells (%DC) and chromosome aberrations (%CA). The expression of p53 was investigated by flow cytometric assay using the monoclonal antibody DO-7, and expressed as percentage positive cells and mean fluorescence intensity. RESULTS: BLM significantly increased both percentage DC and percentage CA and p53 expression (P < 0.01). The %DC was unaffected by the tested peptides. IGF-I [150 microg L(-1)] increased spontaneous percentage CA (P < 0.01). All peptides further increased the BLM-induced chromosome breakage: GH 100 and 1000 microg L(-1) by 30% and 73% respectively, IGF-I 150 and 1000 microg L(-1) by 41% and 96% respectively and IGF-II 600 and 1200 microg L(-1) by 89% and 45% respectively. The spontaneous and BLM-induced expression of p53 was unaffected by GH, whereas it was significantly increased by IGFs (P < 0001). CONCLUSIONS: These results indicate that the DNA-damaging effect of BLM is amplified by GH and, more markedly, IGF-I and -II. IGF-I and -II also stimulate p53 protein expression that, taking part in DNA repair, may counteract the IGF action on genome stability.

Drug-induced changes of carcinoembryonic antigen expression in human cancer cells: effect of 5-fluorouracil.

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.

Differential effects of recombinant interferon-alpha and 5-fluorouracil against colon cancer cells or against peripheral blood mononuclear cells.

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.

Effects of diheptyldiselenide (DDS) on human tumor cell lines and on peripheral blood mononuclear cells.

In vitro effects of graded concentrations of diheptyldiselenide (DDS) on human tumor cell proliferation, and on the proliferative responses and immunological functions of peripheral blood mononuclear cells (MNC) were investigated. The agent significantly decreased tumor cell proliferation in a dose and time dependent manner. Proliferative responses of MNC to phytohemagglutinin (PHA) and interleukin-2 (IL-2) were also significantly depressed when MNCs were exposed to DDS (250 microM for 18 h) led to a significant increase in NK activity only in MNC samples showing very limited baseline NK function. On the other hand, generation of LAK cells was significantly inhibited by DDS. However, when the agent was added to the effector and target cell mixture during the 4 h 51Cr release cytotoxicity assay, no influence was found on NK and LAK-mediated target cell lysis. These studies show that high concentrations of DDS inhibit tumor cell proliferation and could also impair certain proliferative-dependent immune functions, without directly affecting cell-mediated cytolytic activity of effector cells.

Immuno-chemotherapy of advanced colorectal cancer with alpha-2a interferon and 5-fluorouracil. Immunopharmacological studies.

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.

Role of vincristine in immunochemotherapy of leukemia in mouse or human models.

Synergistic antitumor effects between Vincristine (VCR) and allograft responses have been found in mice bearing allogeneic retrovirus-induced leukemia. In this model VCR depressed weakly allograft reactivity if given before but not after antigen administration. In a parallel human tumor model in vitro using HTLV-1 induced MT-2 leukemia, additive but not synergistic immuno-chemotherapeutic effects were obtained with allogeneic mononuclear cells (MNC) combined with VCR at 0.1 but not at 1 micrograms/ml. In this case natural immunity (NI) rather than antigen-dependent immunity (ADI) was involved in the combined effects of VCR + MNC. In the in vitro model pretreatment of effector cells with 1 or 0.1 micrograms/ml of VCR depressed natural cell-mediated cytotoxicity (NCMC). However when the drug was added to the effector + target cells during the 4 h cytotoxicity assay, 1 but not 0.1 micrograms/ml of the drug was capable of depressing NCMC function. These results would provide valuable information for developing in vitro immuno-chemotherapy studies in human tumor systems, including those characterized by the presence of tumor-associated oncogenic retroviruses, capable of depressing both NI and ADI functions.