RESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immune stimulant when administered with different vaccines. Optimal use of GM-CSF resides in its ability to act locally to stimulate the proliferation and maturation of professional antigen-presenting cells (APCs) (i.e., Langerhans' cells) at the injection site. GM-CSF was engineered into a replication-incompetent recombinant avian (fowlpox) virus (rF-GM-CSF) and a single subcutaneous injection resulted in a sustained enrichment of activated dendritic cells within the regional draining lymph nodes. Those changes were attributed to local GM-CSF production at the injection site by rF-GM-CSF-infected cells. Studies were carried out in which mice were administered different types of beta-galactosidase (beta-gal)-based vaccines--whole protein, peptide, recombinant poxviruses--and GM-CSF was administered either as a single injection of rF-GM-CSF or four daily bolus injections of the recombinant protein. The use of rF-GM-CSF either improved the immune adjuvant effect, as observed for poxvirus-based vaccines, or was equivalent to rGM-CSF, as observed with the beta-gal protein vaccine. It is important to note that with either the replication-competent (vaccinia) or replication-incompetent (fowlpox) vaccines expressing LacZ, strong CTL responses directed against beta-gal were induced only when rF-GM-CSF was used as the immune adjuvant. Engineering GM-CSF into a recombinant fowlpox virus offers an excellent vehicle for the delivery of this cytokine as an immune adjuvant with specific vaccine platforms. In particular, delivery of GM-CSF via the rF-GM-CSF construct would be preferred over bolus injections of rGM-CSF when used as an immune adjuvant with whole protein or recombinant poxvirus-based vaccines. The study underscores the importance of defining the appropriate delivery form of an immune adjuvant, such as GM-CSF, relative to the immunization strategy to maximize the host immune responses against a specific antigen.
Assuntos
Avipoxvirus/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Animais , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Camundongos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Linfócitos T Citotóxicos/imunologiaAssuntos
Vacinas Anticâncer/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Afinidade de Anticorpos , Antígenos de Neoplasias/genética , Vacinas Anticâncer/genética , Ensaios Clínicos como Assunto , Epitopos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Humanos , Camundongos , Camundongos TransgênicosRESUMO
Surface-bound IgE play a central role in antiparasite immunity; to exploit IgE-driven immune mechanisms in tumor prevention and control, monoclonal IgEs of irrelevant specificity were loaded through biotin-avidin bridging onto tumor cells, either by systemic administration to tumor-bearing mice or pre-loading of tumor cells before inoculation. Here we show that systemic administration of biotinylated IgEs to mice bearing tumors pre-targeted with biotinylated antibodies and avidin significantly decreased tumor growth rate. In addition, as compared with IgG-loaded control cells, inoculation of suboptimal doses of IgE-loaded tumor cells suppressed tumor formation in a fraction of animals and induced protective host immunity by eliciting tumor-specific T-cell responses. Similarly, tumor vaccination experiments showed that irradiated tumor cells (IgE loaded by biotin-avidin bridging) conferred protective immunity at doses 100-fold lower than the corresponding control cells without IgE. Finally, in vivo depletion of eosinophils or T cells abrogated IgE-driven tumor growth inhibition. These results demonstrate that IgEs targeted on tumor cells not only possess a curative potential but also confer long-term antitumor immunity and that IgE-driven antitumor activity is not restricted to the activation of innate immunity effector mechanisms but also results from eosinophil-dependent priming of a T-cell-mediated adaptive immune response. This suggests a potential role for IgEs in the design of new cell-based tumor vaccines.
Assuntos
Anticorpos Monoclonais/farmacologia , Imunoglobulina E/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , Divisão Celular/efeitos dos fármacos , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Feminino , Interleucina-5/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Análise de Sobrevida , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas/imunologia , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
The use of reverse transcription-PCR (RT-PCR) to analyze cells in the blood of cancer patients for the detection of mRNA expressed in tumor cells has implications for both the prognosis and the monitoring of cancer patients for the efficacy of established or experimental therapies. Carcinoembryonic antigen (CEA) is expressed on approximately 95% of colorectal, gastric, and pancreatic tumors, and on the majority of breast, non-small cell lung, and head and neck carcinomas. CEA shed in serum is useful as a marker in only approximately 50% of colorectal cancer patients and rarely is shed by some other carcinoma types. RT-PCR has been used previously to detect CEA mRNA in cells in the blood and lymph nodes of cancer patients. Under the assay conditions validated in the studies reported here, 34 of 51 (67%) patients with different stages of colorectal cancer had blood cells that were positive by RT-PCR for CEA mRNA, whereas none of 18 patients with colonic polyps were positive; 2 of 60 apparently healthy individuals (who were age and sex matched with the carcinoma patients and were part of a colon cancer screening program as controls) were marginally positive. The results of CEA PCR in the blood of the carcinoma patients and the other groups showed strong statistical correlation with the disease (P2 < 0.0001). Analyses were carried out to detect both serum CEA protein levels and CEA mRNA in blood cells of colorectal carcinoma patients by RT-PCR. For all stages of disease, 18 of 51 patients (35%) were positive for serum CEA, whereas 35 of 51 (69%) were positive by RT-PCR. More importantly, only 5 of 23 (20%) of stage B and C colorectal cancer patients were positive for serum CEA, whereas 16 of 23 (70%) were positive by RT-PCR. The use of two other serum markers (CA19.9 and CA72-4) for colorectal cancer in combination with serum CEA scored two additional patients as positive; both were positive by RT-PCR for CEA mRNA. Pilot long-term longitudinal studies conducted before and after surgery identified some patients with CEA mRNA in blood cells that were negative for all serum markers, who eventually developed clinical metastatic disease. The studies reported here are the first to correlate RT-PCR results for CEA mRNA in blood cells with one or more serum markers for patients with different stages of colorectal cancer, and are the first long-term longitudinal studies to use RT-PCR to detect CEA mRNA in blood cells of cancer patients. Larger cohorts will be required in future studies to define the impact, if any, of this technology on prognosis and/or disease monitoring.
Assuntos
Adenocarcinoma/sangue , Biomarcadores Tumorais/sangue , Antígeno Carcinoembrionário/genética , Neoplasias Colorretais/sangue , Células Neoplásicas Circulantes/imunologia , RNA Mensageiro/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Glicosídicos Associados a Tumores/sangue , Antígenos Glicosídicos Associados a Tumores/genética , Biomarcadores Tumorais/genética , Antígeno CA-19-9/sangue , Antígeno CA-19-9/genética , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/sangue , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Feminino , Humanos , Imuno-Histoquímica , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/metabolismo , RNA Mensageiro/genética , Sensibilidade e EspecificidadeRESUMO
Recombinant avian poxviruses [fowlpox and canarypox (ALVAC)], restricted for replication in nonavian cell substrates and expressing granulocyte/macrophage-colony stimulating factor (avipox-GM-CSF), were evaluated for their ability to enrich an immunization site with antigen-presenting cells (APCs) and, in turn, function as biological vaccine adjuvants. Avipox-GM-CSF administered as a single s.c. injection significantly enhanced the percentage and absolute number of APCs in the regional lymph nodes that drain the injection site. Both the magnitude and duration of the cellular and phenotypic increases within the lymph nodes induced by the avipox-GM-CSF viruses were significantly (P < 0.05) greater than those measured in mice treated with four daily injections of recombinant GM-CSF protein. Temporal studies revealed that the APC enrichment of regional lymph nodes was sustained for 21-28 days after injection of the recombinant avipox virus expressing GM-CSF and, moreover, three injections of the recombinant virus could be given without any appreciable loss of in vivo bioactivity. Mice expressing human carcinoembryonic antigen (CEA) as a transgene (CEA.Tg) developed CEA-specific humoral and cell-mediated immunity after being immunized with avipox-CEA. The coadministration of recombinant avipox viruses expressing CEA and GM-CSF significantly enhanced CEA-specific host immunity with an accompanying immunotherapeutic response in tumor-bearing CEA.Tg mice. The optimal use of avipox-GM-CSF, in terms of dose and dose schedule, especially when used with different immunogens, remains to be determined. Nonetheless, the present findings demonstrate: (a) the effective delivery of GM-CSF to an immunization site using a recombinant avian poxvirus; (b) the compatibility of delivering an antigen and GM-CSF in replication-defective viruses to enhance antigen-specific immunity; and (c) the combined use of recombinant avipox viruses expressing CEA and GM-CSF to generate antitumor immunity directed at a self tumor antigen.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Vacinas Anticâncer/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Linfonodos/imunologia , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/sangue , Avipoxvirus/genética , Vacinas Anticâncer/genética , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Embrião de Galinha , Epitopos de Linfócito T/imunologia , Feminino , Vetores Genéticos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Linfonodos/citologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Proteínas Recombinantes , Linfócitos T/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Antígeno Carcinoembrionário/biossíntese , Fluoruracila/farmacologia , Células HT29/efeitos dos fármacos , Células HT29/imunologia , Apoptose/efeitos dos fármacos , Antígeno Carcinoembrionário/imunologia , Divisão Celular/efeitos dos fármacos , Células Clonais , Células HT29/patologia , HumanosRESUMO
Repeated subcutaneous (s.c.) injections of recombinant granulocyte-macrophage colony-stimulating factor (recGM-CSF) for 4-5 days can enrich an immunization site with antigen-presenting cells (APC), which has been correlated with improved immune responses in experimental and clinical studies. A recombinant vaccinia virus encoding the GM-CSF gene (rV-GM-CSF) has been developed and can generate specific antitumour immunity in a whole tumour cell vaccine. In the present study, we examined whether rV-GM-CSF could produce and release GM-CSF locally which, in turn, might enrich a site of immunization for APC as previously shown for recGM-CSF. S.c. injection of rV-GM-CSF significantly (P<0.05) enhanced the percentage and overall number of APC, measured by class II expression levels, in the regional lymph nodes that drain the injection site. Dose- and temporal-dependent studies showed class II expression levels in the draining lymph nodes were maximally enhanced 5-7 days after a single injection of 10(7)plaque-forming units (pfu) of rV-GM-CSF. Flow cytometry revealed that the increase in class II expression resulted from (i) a higher class II expression level on CD19(+)B cells and (ii) an increase in the number of CD11c(+)/class II(+)professional APC within the draining lymph nodes. Moreover, isolation of lymph nodes from rV-GM-CSF-treated mice revealed their capacity to support higher levels of antigen-specific T cell proliferation and allospecific cytotoxic responses. A comparison between a single injection of rV-GM-CSF and a 4-day course of recGM-CSF revealed comparable changes in class II expression and functional T cell assays. GM-CSF can be delivered in a recombinant poxvirus, and the local production of the cytokine results in cellular and phenotypic changes that are similar to those of recGM-CSF. The ability to utilize rV-GM-CSF as a single inoculum may be more compatible with traditional immunization strategies.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Linfonodos/imunologia , Vaccinia virus , Animais , Células Cultivadas , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Antígenos de Histocompatibilidade Classe II , Imunofenotipagem , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas RecombinantesRESUMO
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.
Assuntos
Antígeno Carcinoembrionário/efeitos dos fármacos , Neoplasias do Colo/patologia , Inibidores Enzimáticos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Estaurosporina/farmacologia , Apoptose , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/genética , Ciclo Celular/efeitos dos fármacos , Neoplasias do Colo/imunologia , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Células HT29/efeitos dos fármacos , Células HT29/imunologia , HumanosRESUMO
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.
Assuntos
Antígeno Carcinoembrionário/metabolismo , Fluoruracila/farmacologia , Interferon gama/farmacologia , Neoplasias Retais/metabolismo , Adulto , Antígeno Carcinoembrionário/sangue , Feminino , Citometria de Fluxo , Humanos , Proteínas Recombinantes , Neoplasias Retais/patologia , Neoplasias Retais/cirurgia , Células Tumorais CultivadasRESUMO
Cytogenetic alterations associated with different stages in carcinogenesis can be distinguished in cultured human or rodent cells transformed by carcinogenic agents. Three tumorigenic rat mammary epithelial cell lines transformed in vitro with 7,12, -dimethylbenz[a]anthracene alone or in combination with 12-O-tetradecanoylphorbol-13-acetate were examined cytogenetically. Non-random alterations consisting of translocations involving the short arm of chromosome 3 and trisomy of chromosomes 14 and X were identified in all three lines. Deletion and inversion of chromosome 1 with the breakpoint at band 1q22 and a duplication 1q 32-43 and trisomy of chromosome 2 were observed in two cell lines. The accumulation of structural alterations and chromosome imbalances during the process of cell immortalization and acquisition of tumorigenicity are required for normal rat mammary cells to become malignant. Unbalanced translocations of chromosome 3 resulting in loss of the short arm had the breakpoint at 3p11. This site is a hotspot of breakage and recombination in various rat tumors and may represent a region of tumor suppressor gene critical to the development of rat mammary tumors, as well as other types of tumors.
Assuntos
Aberrações Cromossômicas , Genes Supressores de Tumor , Neoplasias Mamárias Experimentais/genética , Animais , Linhagem Celular Transformada , Feminino , Humanos , Cariotipagem , Neoplasias Mamárias Experimentais/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
Human carcinoembryonic antigen (CEA) is a well-characterized oncofetal glycoprotein whose overexpression by human carcinomas has been a target for cancer immunotherapy. Transgenic mice that express CEA as a self-antigen with a tissue distribution similar to that of humans have been developed. This study investigates: (a) the responsiveness of the CEA transgenic (CEA.Tg) mice to endogenous CEA or CEA administered as a whole protein in adjuvant; and (b) whether the presentation of CEA as a recombinant vaccinia virus could generate CEA-specific host immunity. By and large, the CEA.Tg mice were unresponsive to CEA, as shown by the lack of detectable CEA-specific serum antibodies and the inability to prime an in vitro splenic T-cell response to CEA. Furthermore, the administration of whole CEA protein in adjuvant to CEA.Tg mice failed to elicit either anti-CEA IgG titers or CEA-specific T-cell responses. Only weak anti-CEA IgM antibody titers were found in those mice. In contrast, CEA.Tg mice immunized with recombinant vaccinia virus expressing CEA generated relatively strong anti-CEA IgG antibody titers and demonstrated evidence of immunoglobulin class switching. These mice also developed T(H)1-type CEA-specific CD4+ responses and CEA peptide-specific cytotoxicity. The ability to generate CEA-specific host immunity correlated with protection of the CEA.Tg mice against a challenge with CEA-expressing tumor cells. Protection against tumor growth was accomplished with no apparent immune response directed at CEA-positive normal tissue. The results demonstrate the ability to generate an effective antitumor immune response to a tumor self-antigen by immunization with a recombinant vaccinia virus. CEA.Tg mice should be an excellent experimental model to study the effects of more aggressive immunization schemes directed at established tumors with the possible development of accompanying autoimmune responses involving normal tissues.
Assuntos
Vacinas Anticâncer/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Neoplasias Experimentais/prevenção & controle , Vaccinia virus/genética , Vaccinia virus/imunologia , Animais , Formação de Anticorpos/imunologia , Especificidade de Anticorpos , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Antígeno Carcinoembrionário/genética , Feminino , Humanos , Imunidade Celular/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinação , Vaccinia virus/metabolismoRESUMO
Surgery, the most effective treatment for colon and rectal cancer, is based on empirical knowledge of the patterns of tumor spread, gross findings at laparotomy, and histologic confirmation of tumor-free margins. In spite of the many technical improvements in surgery, there has not been a significant change in cure rates for colon and rectal cancers. In fact, one-half of affected patients will not survive 5 years. It is in this arena of treatment for primary colon and rectal cancer patients that radioimmunoguided surgery (RIGS) technology may provide the most benefit. RIGS is an intraoperative procedure for detection of carcinoma lesions that are targeted with a radiolabeled monoclonal antibody (MAb) to provide the surgeon with immediate intraoperative definition of tumor margins and identification of occult disease. To optimize this technique, our studies were designed to increase tumor uptake by higher affinity CC-49 (a second-generation MAb) and to increase tumor antigen expression using biological response modifiers (BRMs). The ability of BRMs, such as interferons (IFNs), to enhance the expression of tumor-associated antigens, may play an important role in an adjuvant setting for MAb-based treatment. Preclinical and clinical data provided evidence for the use of IFN as an adjuvant to enhance MAb-targeting of human carcinoma lesions. A combination protocol with IFN and RIGS is ongoing at our institution.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antígenos de Neoplasias/biossíntese , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/cirurgia , Radioimunodetecção , Neoplasias Retais/diagnóstico por imagem , Neoplasias Retais/cirurgia , Antígenos de Neoplasias/imunologia , Antígeno Carcinoembrionário/biossíntese , Neoplasias do Colo/imunologia , Citocinas/farmacologia , Glicoproteínas/biossíntese , Humanos , Imuno-Histoquímica , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Estadiamento de Neoplasias , Radioisótopos , Proteínas Recombinantes , Neoplasias Retais/imunologiaRESUMO
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.
Assuntos
Antígeno Carcinoembrionário/análise , Neoplasias do Colo/tratamento farmacológico , Fluoruracila/administração & dosagem , Interferon gama/administração & dosagem , Leucovorina/administração & dosagem , Animais , Antígeno B7-1/análise , Western Blotting , Antígeno Carcinoembrionário/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Feminino , Células HT29 , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , RNA Mensageiro/análise , Transplante HeterólogoRESUMO
A recombinant vaccinia virus encoding the gene for granulocyte-macrophage colony-stimulating factor (rV-GM-CSF) was used to infect the poorly immunogenic murine colon adenocarcinoma cell line, MC-38. Infection of MC-38 tumor cells with rV-GM-CSF completely suppressed the growth of the MC-38 primary tumors, whereas progressively growing tumors were formed in mice injected with MC-38 cells infected with wild type V-Wyeth. Irradiation of the recipient B6 mice before implantation of rV-GM-CSF-infected tumor cells resulted in the development of progressively growing tumors. Moreover, in vivo T-cell depletion studies revealed that growth suppression of the rV-GM-CSF-infected tumor cells was dependent on the presence of both CD4+ and CD8+ T-cell subsets. Subsequent studies established that this immunity was long-lasting and antigen specific, as demonstrated by the protection of rV-GM-CSF-immunized mice from MC-38 tumor challenge but not from challenge with another syngeneic tumor cell type. No such effects were observed when MC-38 tumor cells were infected with recombinant vaccinia viruses expressing interleukin (IL)-2 or IL-6. The results demonstrate that paracrine release of biologically active murine GM-CSF by tumor cells infected with rV-GM-CSF enhances the intrinsic immunogenicity of a poorly immunogenic murine tumor. Presumably the augmentation of tumor immunogenicity induces an antigen-specific T-cell-dependent antitumor response that prevents the formation of primary tumors and protects mice from tumor challenge. Thus in this experimental model, GM-CSF functions as a highly effective vaccine adjuvant.
Assuntos
Adenocarcinoma/imunologia , Vacinas Anticâncer , Neoplasias do Colo/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Imunização , Vaccinia virus/genética , Adenocarcinoma/prevenção & controle , Animais , Linhagem Celular , Neoplasias do Colo/prevenção & controle , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Haplorrinos , Rim , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Proteínas Recombinantes , Células Tumorais CultivadasRESUMO
Tissues and sera from 110 patients diagnosed with colorectal primary carcinoma, 20 patients with benign colorectal diseases and 31 healthy donors were subjected to quantitative CEA analysis. Multiple samples from tumor lesions and autologous histologically normal mucosa (10 cm from the tumor) were obtained at the time of surgery (cancer patients) or endoscopy (benign patients and healthy volunteers). CEA content was measured in protein extracts obtained from these tissues using a quantitative RIA method. A limit of normality for CEA content was established as 300 ng/mg of protein. When this was taken as cut-off, 104 of 110 (94.5%) tumor lesions and 51 of 110 (46.4%) autologous histologically normal colonic mucosa from cancer patients had elevated CEA levels. No correlation with stage of disease was found, while a correlation was observed with degree of tumor differentiation. A statistically significant difference between CEA content in tumor lesions and in histologically normal mucosa from cancer patients was observed (p = -0.001). Moreover, CEA content was statistically higher in the normal mucosa from cancer patients than in that from healthy donors (p = 0.005). CEA content in tissue specimens from benign lesions differed significantly from that in tissue from healthy donors (p = 0.005) and in carcinoma lesions (p < 0.001). The highest CEA content was observed in benign lesions with severe dysplasia. No statistical correlation between CEA content in carcinoma tissues and serum CEA levels (r = 0.195, p = .13) was found. Therefore, in considering diagnosis or therapy with anti-CEA MAbs for colorectal-carcinoma patients, or potential therapies with anti-CEA recombinant vaccines, serum CEA levels should not be taken as indicating CEA expression in tumor lesions.
Assuntos
Adenocarcinoma/química , Antígeno Carcinoembrionário/análise , Neoplasias Colorretais/química , Mucosa Intestinal/química , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Antígeno Carcinoembrionário/sangue , Colo/citologia , Colo/patologia , Doenças do Colo/patologia , Neoplasias Colorretais/patologia , Feminino , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Radioimunoensaio , Valores de Referência , Análise de RegressãoRESUMO
Strong immunogenicity is induced by antitumor triazene compounds in tumor cells through a mutagenic mechanism. A highly immunogenic <
Assuntos
Antineoplásicos/farmacologia , Dacarbazina/farmacologia , Genes ras/imunologia , Leucemia L5178/imunologia , Transfecção , Animais , Genes ras/genética , Antígenos de Histocompatibilidade Classe I/análise , Leucemia L5178/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Transfecção/genéticaRESUMO
Preclinical studies have demonstrated that recombinant IFN-alpha (rIFN-alpha) can enhance the tumor associated glycoprotein 72 (TAG-72) on tumors. To determine whether rIFN-alpha could enhance TAG-72 expression in vivo in patients, 15 women with breast cancer were randomized to receive daily injections of rIFN-alpha (3 x 10(6) units/m2 for 14 days) beginning on day 1 (group 1 = 7 patients) or on day 6 (group 2 = 8 patients). On day 3, all patients received a 10-20-mCi tracer dose of 131I-CC49, a high-affinity murine monoclonal antibody reactive against TAG-72, followed by a therapy dose of 60-75 mCi/m2 of 131I-CC49 on day 6. Whole body and single-photon emission computed tomography scans along with whole blood pharmacokinetics were performed following tracer and treatment phases. Hematological toxicity was considerable; reversible grade 3-4 neutropenia and thrombocytopenia was observed in 12 of 15 patients. Twelve of 14 patients tested developed human antimouse antibodies 3-6 weeks after treatment. For group 1 patients, whole blood residence time increased significantly between that predicted from the tracer doses and therapy doses (42.6 +/- 4.7 versus 51.5 +/- 4.8 h, respectively; P < 0.01). The calculated radiation absorbed dose to red marrow from therapy compared to tracer activity was also significantly higher for this group (1.25 +/- 0.35 versus 1. 07 +/- 0.26 cGy/mCi; P < 0.05). Treatment with rIFN-alpha was found to enhance TAG-72 expression in tumors from patients receiving rIFN-alpha (group 1) by 46 +/- 19% (P < 0.05) compared to only 1.3 +/- 0.95% in patients not initially receiving IFN (group 2). The uptake of CC49 in tumors was also significantly increased in rIFN-alpha-treated patients. One partial and two minor tumor responses were seen. In summary, rIFN-alpha treatment altered the pharmacokinetics and tumor uptake of 131I-CC49 in patients at the expense of increased toxicity.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Glicoproteínas/imunologia , Imunoconjugados/farmacocinética , Interferon-alfa/farmacologia , Radioisótopos do Iodo/farmacocinética , Radioimunoterapia , Adulto , Animais , Anticorpos Anti-Idiotípicos/biossíntese , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Medula Óssea/efeitos da radiação , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/imunologia , Neoplasias da Mama/radioterapia , Terapia Combinada , Esquema de Medicação , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/biossíntese , Glicoproteínas/genética , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/uso terapêutico , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Interferon-alfa/uso terapêutico , Radioisótopos do Iodo/efeitos adversos , Radioisótopos do Iodo/uso terapêutico , Metástase Linfática/radioterapia , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Neutropenia/induzido quimicamente , Proteínas Recombinantes , Trombocitopenia/induzido quimicamente , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Resultado do TratamentoRESUMO
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.
Assuntos
Antígeno Carcinoembrionário/genética , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , Citometria de Fluxo , Humanos , Células Tumorais CultivadasRESUMO
Colorectal tissue biopsies were obtained from 110 patients diagnosed with primary colorectal carcinoma (tumor and normal colonic mucosa samples), 20 patients diagnosed with benign colorectal disease, and 31 healthy donors. The level of expression of tumor-associated glycoprotein 72 (TAG-72) was quantitatively measured in each sample using a double-determinant RIA with monoclonal antibodies B72.3 and CC49 and detecting the sialyl-Tn epitope; this assay was termed CA 72-4. Statistical analysis revealed a significant (approximately 10-fold) increase of TAG-72 expression in the colon tumor biopsies when compared with the expression in normal colonic mucosa from the same patients. A regression analysis revealed a significant correlation (r = 0.459; P < 0.001) between TAG-72 levels measured in biopsies from the tumor lesions and those found in the corresponding normal colonic mucosa. Furthermore, regression analysis showed a significant positive correlation between TAG-72 levels in the tumors and sera of the same patients (r = 0.491; P < 0.001). TAG-72 levels in normal colonic mucosa from healthy donors and patients diagnosed with colorectal cancer were compared. TAG-72 expression was 5-fold higher in the normal mucosa from the colorectal carcinoma patients. No relationship between TAG-72 tumor tissue content and stage of disease was found. Moreover, the correlation between TAG-72 distribution and degree of tumor differentiation observed (P < 0.05) was not any more evident when mucinous carcinomas were excluded. Finally, the results provide further evidence that TAG-72 may be considered an important early marker for colorectal cancer and/or other dysplastic colonic diseases. The statistical correlation between TAG-72 levels in tumors and circulating TAG-72 indicates that patients with elevated levels of serum TAG-72, as measured by the CA 72-4 assay, would be most suited for diagnostic and/or therapeutic intervention with the anti-TAG-72 monoclonal antibodies B72.3 or CC49 or vaccine trials using the sialyl-Tn epitope.
Assuntos
Antígenos de Neoplasias/análise , Neoplasias Colorretais/química , Glicoproteínas/análise , Proteínas de Neoplasias/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/sangue , Neoplasias Colorretais/sangue , Feminino , Glicoproteínas/sangue , Humanos , Mucosa Intestinal/química , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/sangueRESUMO
Cytokine-based tumor antigen augmentation is one of the approaches researchers and clinicians are using to improve the effectiveness of MAb-directed tumor diagnosis and therapy. Other efforts encompass the use of dose-fractionation for multiple administrations of radioimmunoconjugates, exploitation of genetic engineering to construct antibody molecules with specific biological properties (i.e., altered pharmacokinetics, activation of cellular immune responses, etc.) and use of MAb-directed conjugates that can enhance tumor MAb uptake by altering tumor perfusion. The studies summarized here as well as those from other laboratories have served as the framework for clinical investigations designed to determine the effectiveness of the interferons and other differentiation-inducing agents to alter the tumor antigen phenotype in patients. In an earlier study, patients given IFN-alpha had improved tumor uptake of an antimelanoma MAb. Subsequently, we reported that i.p. IFN-gamma administration substantially upregulated TAG-72 and CEA on the surface of human tumor cells isolated from malignant ascites. A seminal investigation showed significant increase of TAG-72 and CEA levels in tumor biopsies from patients diagnosed with colorectal carcinoma and given systemic IFN-alpha. Those studies led to a clinical trial in which late stage breast cancer patients were administered interferon in combination with therapeutic doses of CC49. Some clinical responses were observed, however, the cytokine and MAb combination may have also enhanced marrow toxicity. Future studies will continue to evaluate the ability to enhance tumor antigen expression in the context of genetically engineered MAbs designed to minimize normal organ toxicity.