RESUMEN
The combination of N-(phosphonacetyl)-L-aspartate (PALA), 6-methylmercaptopurine riboside (MMPR), and 6-aminonicotinamide (6AN) has been shown to be an effective antineoplastic regimen and also to enhance the effects of other antineoplastic agents (1-4). To further enhance the effect of this combination, we investigated the effects of adding adriamycin, at its maximally tolerated dose, to this regimen. The response rate (complete regression+partial regression) for the four-drug regimen was higher than for the three-drug regimen, and the tumor growth delay was also significantly higher than for treatment with PALA, MMPR, 6AN, or after treatment with maximally tolerated doses of adriamycin alone (11 mg/kg). The addition of adriamycin to PALA, MMPR, 6AN did not result in enhancement of the effect of radiation, as measured by tumor growth delay studies and tumor control (complete and partial regression rate). The mechanism of action of the combination of PALA, MMPR, and 6AN is not known definitively, but a possible mechanism previously suggested is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Treatment with PALA, MMPR, 6AN, and adriamycin (at 2.5 hr post MMPR, 6AN) resulted in a nadir NTP/Pi value, as determined by 31P NMR spectroscopy, at approximately 10 hr post MMPR + 6AN (7.5 hr post adriamycin), which was not significantly different from the NTP/Pi value determined after treatment with the three-drug combination.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Espectroscopía de Resonancia Magnética/métodos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/radioterapia , Fármacos Sensibilizantes a Radiaciones/farmacología , 6-Aminonicotinamida/administración & dosificación , Animales , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , División Celular/efectos de los fármacos , Progresión de la Enfermedad , Doxorrubicina/administración & dosificación , Sinergismo Farmacológico , Estudios de Evaluación como Asunto , Metiltioinosina/administración & dosificación , Ratones , Isótopos de FósforoRESUMEN
The drug combination N-(phosphonacetyl)-L-aspartic acid (PALA), methylmercaptopurine riboside (MMPR) and 6-aminonicotinamide (6AN), referred to as PMA, induces regressions of advanced CD8F1 murine mammary carcinomas in vivo. We demonstrated that CD8F1 tumor regressions were preceded by the appearance of apoptotic bodies, as observed by microscopic examination of morphology and TUNEL endlabeling, and fragmentation of DNA into nucleosomal "ladder" patterns. These indications of apoptosis were present as early as 6 h after simultaneous administration of MMPR and 6AN and further increased by over fivefold during the next 3 to 6 h, then remained at 7 to 12.8% (0.6 to 2.4% in saline-treated controls) of the cell population for at least 24 h after MMPR + 6AN administration. The 5'-phosphate derivative of MMRP, MMPR-5P, which inhibits de novo purine biosynthesis, was present at a "steady-state" level, and significant (40%) depletion of ATP had occurred by 3 h and both of these events preceded the onset of apoptosis. In addition, MMPR-5P was retained in CD8F1 tumors at a high level over a prolonged period (> 96 h) even as tumors were undergoing regression. The prolonged presence of MMPR-5P was important for optimal chemotherapeutic effect, since treatment with iodotubercidin (IodoT), an inhibitor of MMPR/adenosine kinase, 6 h after MMPR+6AN administration prevented the prolonged accumulation of MMPR-5P and reversed the regression of CD8F1 tumors. In addition, compared to the PMA-treated group, there was a significant restoration of ATP levels after treatment with IodoT. In individual PMA-treated CD8F1 tumors the degree of ATP depletion was found to correlate with the degree of tumor shrinkage at 24 h, after tumors had sufficient time to respond to treatment. These results define the time-course of drug-induced apoptosis in CD8F1 tumors, show that ATP depletion occurs prior to apoptosis and demonstrate that prolonged retention of MMPR-5P is associated with optimal chemotherapy. Collectively, these results suggest that the depletion of ATP by PMA treatment may be a component of the biochemical apoptotic cascade in the CD8F1 tumor.
Asunto(s)
Adenosina Trifosfato/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , 6-Aminonicotinamida/administración & dosificación , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Femenino , Mercaptopurina/administración & dosificación , Mercaptopurina/análogos & derivados , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivados , Factores de TiempoRESUMEN
Paclitaxel alone is active against the CD8F1 murine spontaneous mammary cancer, and when administered following an ATP-depleting combination of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6-AN) (PMA) produced significantly enhanced partial tumor regressions over that produced by either paclitaxel alone at the maximal tolerated dose (MTD), or by the PMA drug combination alone, against advanced, first passage spontaneous murine breast tumors. The anticancer activity of paclitaxel is due to enhancement and stabilization of microtubule polymerization. Pertinently, microtubule disassembly (an ATP-dependent process) is known to sharply decrease in the presence of ATP depletion. Thus, the dramatic therapeutic enhancement observed with paclitaxel in combination with PMA is in agreement with biochemical expectations, since PMA has been shown to deplete ATP in CD8F1 tumor cells. The augmented therapeutic results were obtained with approximately one-third the MTD of paclitaxel as a single agent and suggest the potential clinical benefit of more effective treatment with lesser amounts of drug.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Paclitaxel/uso terapéutico , 6-Aminonicotinamida/administración & dosificación , Animales , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Femenino , Metiltioinosina/administración & dosificación , Ratones , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivados , Inducción de RemisiónRESUMEN
Treatment with a combination (PMA) of (N-phosphonacetyl)-L-aspartic acid (PALA), methylmercaptopurine riboside (MMPR), and 6-aminonicotinamide (6AN) induced partial regressions of CD8F1 murine mammary tumors and provided for tumor growth inhibition without regression of Colon 38 tumors. HPLC-nucleotide pool analysis of CD8 mammary tumors obtained at various times after treatment with PMA revealed that MMPR-5'-phosphate, which inhibits de novo purine nucleotide biosynthesis, was constant at levels of approximately 2.5 nmol/mg protein for 72 hr after treatment. In contrast, the MMPR-5'-phosphate levels of C38 tumors decreased from 24-hr levels at 1.5 nmol/mg protein with a half-time of about 24 hr. Treatment of CD8 tumor-bearing mice with iodotubercidin, a potent inhibitor of adenosine/MMPR kinase, at various times after PMA, reversed both the accumulation of high levels of MMPR-5'-phosphate and the number of partial tumor regressions. These data demonstrate that a cycle of MMPR rephosphorylation is active in the CD8 mammary tumor and suggest that this recycling of MMPR is important for the optimal effect of PMA treatment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Metiltioinosina/metabolismo , 6-Aminonicotinamida/administración & dosificación , Adenosina Quinasa/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Neoplasias Mamarias Experimentales/metabolismo , Metiltioinosina/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivadosRESUMEN
A three-drug combination, PMA, consisting of (phosphonacetyl)-L-aspartic acid + 6-methylmercaptopurine riboside + 5-aminonicotinamide, preceding either 5-fluorouracil (5-FU) or adriamycin (Adr), produced tumor-regressing activity in a murine advanced breast tumor model not attainable with either 5-FU or Adr as single agents, or with any lesser combination of these drugs administered at maximally tolerated doses. Marked tumor-regressing activity was further increased significantly by using 5-FU and Adr together in conjunction with the modulatory biochemical conditioning (particularly ATP depletion) provided by pretreatment with PMA.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/administración & dosificación , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Doxorrubicina/administración & dosificación , Fluorouracilo/administración & dosificación , Neoplasias Mamarias Animales/tratamiento farmacológico , Mercaptopurina/administración & dosificación , Mercaptopurina/análogos & derivados , Ratones , Niacinamida/administración & dosificación , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivadosRESUMEN
DNA-damaging agents, e.g. Adriamycin (ADR), are reported to cause tumor regression by induction of apoptosis. A reduction in the intracellular content of ATP is part of the biochemical cascade of events that ultimately results in programmed death of the cell, or apoptosis. A chemotherapeutic three-drug combination (PMA) consisting of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6AN) significantly lowers levels of ATP in CD8F1 murine breast tumors in vivo and produces tumor regression by apoptosis. Addition of the DNA-damaging antitumor agent ADR to PMA was found to further significantly deplete ATP in CD8F1 murine breast tumors in vivo with a concomitant significant increase in the number of tumor regressions. The correlative biochemical and therapeutic results are consistent with, and support, the hypothesis that ATP depletion is a significant factor and, therefore, is a worthy therapeutic target in the production of apoptosis.
Asunto(s)
Adenosina Trifosfato/análisis , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/patología , 6-Aminonicotinamida/administración & dosificación , Animales , Apoptosis , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Doxorrubicina/administración & dosificación , Neoplasias Mamarias Animales/metabolismo , Metiltioinosina/administración & dosificación , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivados , Factores de TiempoRESUMEN
A quadruple drug combination--consisting of a triple-drug combination of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-amino-nicotinamide (6-AN), designed to primarily deplete cellular energy in tumor cells, + Adriamycin (Adria)--yielded significantly enhanced anticancer activity (i.e., tumor regressions) over that produced by either Adria alone at maximum tolerated dose (MTD) or by the triple-drug combination, against large, spontaneous, autochthonous murine breast tumors. The adenosine triphosphate (ATP)-depleting triple-drug combination administered prior to Adria resulted in a 100% tumor regression rate (12% complete regression; 88% partial regression) of spontaneous tumors. Histological examination of treated tumors demonstrated that the treatment-induced mechanism of cancer cell death was by apoptosis. The augmented therapeutic results (100% tumor regressions) were obtained with approximately one-half the MTD of Adria as a single agent and suggest the potential clinical benefit of longer, more effective, and safer treatment by low doses of Adria when combined with the triple-drug combination. Two likely mechanisms of action are discussed: (1) prevention of DNA repair; (2) complementary disruption of biochemical pathways by both the triple-drug combination and the biochemical cascade of apoptosis that is induced by a DNA-damaging anticancer agents such as Adria.
Asunto(s)
Doxorrubicina/administración & dosificación , Neoplasias Mamarias Experimentales/tratamiento farmacológico , 6-Aminonicotinamida/administración & dosificación , Animales , Apoptosis , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Metabolismo Energético , Neoplasias Mamarias Experimentales/metabolismo , Metiltioinosina/administración & dosificación , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivadosRESUMEN
The combination of N-(phosphonacetyl)-L-aspartate, 6-methylmercaptopurine, and 6-aminonicotinamide has been shown to be an effective antineoplastic regimen and also to enhance the effects of other chemotherapeutic agents. The mechanism of action of this combination of drugs is not known definitively, but one possible mechanism is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Tumor-bearing mice were treated with N-(phosphonacetyl)-L-aspartate, followed 17 h later by 6-methylmercaptopurine and 6-aminonicotinamide. 31P nuclear magnetic resonance spectroscopic studies demonstrated a significant depletion of high energy phosphates at 10 h post-6-methylmercaptopurine and 6-aminonicotinamide. The addition of radiation at this time was shown to induce a significantly longer tumor growth delay and a greater number of regressions (including durable complete regressions) than either chemotherapy or radiation alone. The combination of chemotherapy and radiation was found to be supra-additive compared to the antineoplastic effects of either modality administered separately, without a measurable increase in host toxicity.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Experimentales/terapia , 6-Aminonicotinamida/administración & dosificación , Animales , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Terapia Combinada , Femenino , Mercaptopurina/administración & dosificación , Mercaptopurina/análogos & derivados , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Trasplante de Neoplasias , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/radioterapia , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivadosRESUMEN
This report describes a highly active chemotherapeutic drug combination, consisting of N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide plus 5-fluorouracil, in CD8F1 mice bearing spontaneous, autochthonous, breast tumors or first-passage advanced transplants of these spontaneous tumors. The combination and sequence of administration of these drugs were selected on the basis of known potentiating biochemical interactions. High performance liquid chromatography and nuclear magnetic resonance spectroscopy measurements of biochemical changes resulting from treatment with N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide indicated a severe depletion of cellular energy levels in the treated tumors. 6-Aminonicotinamide produced a severe block of the pentose shunt, and 5-fluorouracil severely inhibited both thymidylate synthase and thymidine kinase in the treated tumors. This quadruple drug combination, administered on a 10-11-day schedule, produced an impressive partial tumor regression rate of 67% of large, spontaneous, autochthonous, murine breast tumors and a tumor regression rate of 74% of first-passage transplants of the spontaneous breast tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Fluorouracilo/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , 6-Aminonicotinamida/administración & dosificación , Animales , Antineoplásicos/administración & dosificación , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Femenino , Fluorouracilo/administración & dosificación , Neoplasias Mamarias Experimentales/metabolismo , Metiltioinosina/administración & dosificación , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Ácido Orótico/metabolismo , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivados , Fosforribosil Pirofosfato/metabolismoRESUMEN
Two strategies for modulation of 5-fluorouracil (FUra) activity were compared in vivo in advanced murine CD8F1 breast tumors with regard to three parameters: chemotherapeutic activity, inhibition of thymidylate synthase (TSase) activity, and incorporation of FUra into RNA, (FU)RNA. Inhibition of TSase by FUra was modulated by leucovorin (LV), and the incorporation of FUra into RNA was increased by the administration of otherwise lethal doses of FUra followed by uridine "rescue". Thymidylate synthase activity was inhibited substantially (49%) by low-dose FUra at 25 mg/kg, but was not further enhanced (48%) by repeated daily treatments at the same dose (FUra25 x 4). Inhibition of TSase was somewhat enhanced (55%) by the addition of LV to FUra25 x 4, and a greater therapeutic effect was obtained with FUra25 x 4 + LV over FUra25 x 4 alone. FUra as a single agent at the maximum tolerated weekly dose of 100 mg/kg inhibited TSase activity 66-73%. This inhibition was further enhanced slightly by the addition of LV (71-82%), and its therapeutic efficacy was greater than with FUra25 x 4 with or without LV. However, in contrast to low dose FUra25 x 4, the antitumor effect of FUra100 was not enhanced by LV. (FU)RNA increased with FUra dose from 0.4 (FUra25) to 2.2 nmol/mg DNA (FUra100). At a very-high-dose of FUra (200-225 mg/kg) followed by uridine "rescue", TSase inhibition was not further enhanced, but both (FU)RNA (4.8 nmol/mg DNA) and the therapeutic efficacy were increased. Since TSase could not be further inhibited at doses above FUra100, the increased chemotherapeutic efficacy correlated with increased (FU)RNA.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Fluorouracilo/uso terapéutico , Leucovorina/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Uridina/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fluorouracilo/administración & dosificación , Fluorouracilo/metabolismo , Leucovorina/administración & dosificación , Neoplasias Mamarias Experimentales/patología , Ratones , ARN/metabolismo , Timidilato Sintasa/antagonistas & inhibidores , Factores de TiempoRESUMEN
We have used in vivo 19F NMR spectroscopy to study the metabolism of 5-fluorouracil (FUra) in tumors with and without pretreatment with methotrexate (MTX). Using the CD8F1 murine mammary tumor as an in vivo model, we observed signals from FUra, alpha-fluoro-beta-alanine (F beta ALA), alpha-fluoro-beta-ureidopropionic acid (FUPA), and 5-fluorouracil-nucleotides (FUN) after intravenous or intraperitoneal injection of 150 mg/kg FUra. Formation of FUN was increased about 1.7-fold in CD8F1 tumor with methotrexate pretreatment as determined by acid extraction and HPLC analysis. A comparison of in vivo NMR spectra from FUra and sequential MTX + FUra-treated tumors showed a significantly higher ratio of the FUN signal to the initial total 19F signal in the MTX + FUra-treated tumors (p less than 0.001) for animals receiving FUra either intravenously or intraperitoneally. In addition, tumors treated with MTX + FUra had significantly longer time durations during which FUN was detected, independent of the mode of administration. These experiments indicate that in vivo 19F NMR spectroscopy can be used to noninvasively monitor alterations of 5-fluorouracil metabolism that occur with administration of modulating agents such as methotrexate. Further studies, in both murine tumor models and patients, are indicated to determine if these results can be correlated with tumor response.
Asunto(s)
Fluorouracilo/metabolismo , Espectroscopía de Resonancia Magnética , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Metotrexato/uso terapéutico , Animales , Femenino , Fluorouracilo/uso terapéutico , Masculino , Ratones , PremedicaciónRESUMEN
Most cytotoxic drugs used in cancer therapy do not discriminate between neoplastic and normal proliferating cells. To avoid irreversible damage to vital host tissues, such as bone marrow and intestine, drugs must be administered at dosages which usually prove insufficient to eradicate all of the neoplastic cells present. This review focuses on an approach to improve cancer chemotherapy by selectively protecting normal, proliferating cells during treatment, thereby permitting the administration of otherwise lethal doses of drug. Preclinical in vivo studies of cytokinetic modulation with interferon, or L-histidinol, as well as recent clinical studies of interferon modulation of the activity of 5-fluorouracil are reviewed.
Asunto(s)
Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Sinergismo Farmacológico , Histidinol/farmacología , Histidinol/uso terapéutico , Humanos , Interferones/farmacología , Interferones/uso terapéutico , Neoplasias/patologíaRESUMEN
Myelosuppression following intensive chemotherapy in cancer patients is associated with increased morbidity and mortality. Hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), alone or in combination with interleukin-1 (IL-1), have been shown to counteract myelosuppression resulting from some, but not all, chemotherapeutic regimens. In an attempt to apply these findings to intensive therapy with proliferation-dependent chemotherapeutic drugs such as fluorouracil (5-FU), we investigated combination biochemotherapy in a murine model. Female CD8F1 [(BALB/c X DBA/8)F1] mice bearing first-passage transplants of spontaneous CD8F1 breast tumors were treated intraperitoneally once a week for 3 successive weeks with a course of 5-FU alone or with a course of 5-FU in combination with recombinant human interleukin-1 beta (rHuIL-1 beta) alone or in combination with CSFs. rHuIL-1 beta alone or in combination with rHuG-CSF or recombinant murine GM-CSF significantly improved tumor growth inhibition (60% vs. 90%) and survival (20% vs. 90%-100%), increased the maximally tolerated dose of 5-FU, accelerated recovery of neutrophil counts in peripheral blood, and reduced duration of significant neutropenia and loss of body weight (29% vs. 10% loss). Clinical trials of IL-1 have been initiated in patients with advanced cancer receiving multiple courses of high-dose 5-FU.
Asunto(s)
Médula Ósea/efectos de los fármacos , Factores Estimulantes de Colonias/farmacología , Fluorouracilo/toxicidad , Sustancias de Crecimiento/farmacología , Interleucina-1/farmacología , Animales , Femenino , Factor Estimulante de Colonias de Granulocitos , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Recuento de Leucocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Neoplasias Experimentales/sangre , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Proteínas Recombinantes/farmacologíaRESUMEN
Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth in vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogen- or prolactin-dependent growth. Primary tumors still displayed a constitutional expression of alpha-, beta-, and gamma-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in alpha- and beta-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of alpha-, beta-, and gamma-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10-14 days. The accumulation of beta-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance beta-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.
Asunto(s)
Adenocarcinoma/metabolismo , Caseínas/genética , Estradiol/farmacología , Expresión Génica/efectos de los fármacos , Neoplasias Mamarias Experimentales/metabolismo , Prolactina/farmacología , Adenocarcinoma/patología , Animales , División Celular/efectos de los fármacos , ADN/biosíntesis , Hidrocortisona/farmacología , Neoplasias Mamarias Experimentales/patología , Virus del Tumor Mamario del Ratón , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Trasplante de Neoplasias , ARN Mensajero/biosíntesis , Células Tumorales CultivadasRESUMEN
In experiments designed to investigate the biochemical basis for the diminution of the antitumor activity of 5-fluorouracil (FUra) by L-histidinol in CD8FI breast tumors, it was discovered that L-histidinol inhibits RNA and DNA synthesis in these tumors. This finding suggested the possibility that L-histidinol might have antiproliferative activity in the CD8FI breast tumor, and on that basis we evaluated different administration schedules of L-histidinol for antitumor activity in vivo. The present report describes a schedule of L-histidinol administration which yielded significant activity against spontaneous, autochthonous CD8FI breast tumors consistently at tolerable doses. To our knowledge, there are no previous reports of in vivo antitumor activity associated with the administration of L-histidinol as a single agent.
Asunto(s)
Histidinol/uso terapéutico , Neoplasias Mamarias Animales/tratamiento farmacológico , Animales , ADN de Neoplasias/biosíntesis , Femenino , Fluorouracilo/metabolismo , Ratones , ARN Neoplásico/biosíntesisRESUMEN
This study describes a serum-free culture system and provides a tumor model to investigate the effects of extracellular matrices on the growth and beta-casein gene expression of mouse mammary tumor epithelial cells (MMTCs) in vitro. Primary cultures of MMTCs derived from autochthonous mammary tumors in BALB/cfC3H x DBA/8 F1 mice, FUKU cells, an established MMTC line, and COMMA-1D cells established from mouse mammary tissues were studied. A reconstituted basement membrane from the Englbreth-Holm-Swarm tumor (Matrigel) allowed a 2.7-fold increase in cell number of 5-day primary MMTC cultures in serum-free, insulin-supplemented medium. FUKU and COMMA-1D cells in serum-free medium displayed a 13.6- and 11.5-fold increase in cell number, respectively, after 5 and 6 days in culture on Matrigel. In semisolid agar cultures, Matrigel or laminin was shown to promote colony-forming efficiency of FUKU cells when either of the matrices was mixed in the top agar layer. An increase of 4.4 or 2.1 times in colony-forming efficiency was detected when 20% (v/v) Matrigel or 112 micrograms/ml of laminin were mixed in the agar layer compared with FUKU cells plated in plain agar. beta-Casein mRNA was detectable by Northern blot assays in the primary mammary tumors. MMTCs in primary cultures grown on Matrigel in serum-free, insulin-supplemented medium for 4 days were inducible for beta-casein mRNA following the treatment with prolactin and hydrocortisone (FPRL) for 24 h. No beta-casein mRNA was detectable in the absence of FPRL. MMTCs in the primary cultures could also be induced for beta-casein mRNA when they were cultivated on type I collagen gels for 4 days but not on laminin, type IV collagen, or plastic. However, the capacity to respond to FPRL was not lost in MMTCs cultured on laminin. When MMTCs were initially cultured on laminin for 4 days and then subcultured on Matrigel for another 4 days, they were inducible for beta-casein mRNA upon exposure to FPRL for 24 h. In contrast, no beta-casein mRNA upon exposure to FPRL for 24 h. In contrast, no beta-casein mRNA was found in MMTCs from the same tumors cultured on laminin for 8 days with the same treatment of hormones. These data demonstrate that cells from autochthonous mammary tumors, which are not dependent on estrogen for growth in vivo, are inducible in vitro for beta-casein mRNA by FPRL; and this hormonal response of MMCTs requires appropriate extracellular matrix.
Asunto(s)
Caseínas/genética , Matriz Extracelular , Neoplasias Mamarias Experimentales/patología , Animales , Adhesión Celular , Recuento de Células , División Celular , Colágeno/farmacología , Medios de Cultivo/farmacología , Regulación de la Expresión Génica , Laminina/farmacología , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/metabolismo , Factores de Tiempo , Células Tumorales Cultivadas/patología , Ensayo de Tumor de Célula MadreRESUMEN
As a consequence of the inhibition of de novo purine synthesis by methotrexate (MTX) there is an increase in 5-phosphoribosyl-1-pyrophosphate (PRPP) concentration. In cells where 5-fluorouracil (FUra) is activated via orotate phosphoribosyltransferase (OPRtase), increased PRPP results in greater conversion of FUra to nucleotides. In the murine CD8F1 breast tumor system, MTX markedly enhances the antitumor activity of FUra, increasing both the activation of FUra to FUMP and the incorporation of FUTP into RNA. However, in contrast to reported tumor tissue culture studies, MTX pretreatment in vivo prevents the stable incorporation of FUra into CD8F1 bone marrow DNA. Pretreatment with MTX (300 mg/kg) 2.5 hr prior to [3H]FUra (100 mg/kg), with a 2-hr labeling, reduced the level of FUra in DNA from 921 pmol to 66 pmol/mg of DNA. Without MTX pretreatment, 59% of the total incorporation of FUra into nucleic acids was into DNA when FUra was administered. After MTX the percentage of incorporation into DNA was reduced to 9%. Additionally, the ratio of [3H]FUra to 32P in DNA when both were given simultaneously was reduced by greater than 90%, suggesting that MTX must be specifically blocking the incorporation of FUra rather than nonspecifically preventing its incorporation by inhibiting DNA synthesis. In contrast, MTX failed to reduce the formation of DNA containing fluorouracil residues from FdUrd. To test whether MTX prevents the initial incorporation of FUra into DNA, or acts to enhance removal by the DNA glycosylase repair system, DNA was prelabeled in vivo with [3H]FUra, and MTX or MTX plus dThd was then administered. The level of FUra in bone marrow DNA was not reduced by subsequent treatment with MTX, or MTX plus dThd, indicating that MTX does not enhance the removal of FUra from DNA. The level of total free fluorodeoxynucleotides formed from FUra was reduced by two-thirds following MTX pretreatment, suggesting that the action of MTX in preventing the stable incorporation of FUra into DNA was to reduce the availability of FdUTP.
Asunto(s)
Médula Ósea/metabolismo , ADN/metabolismo , Fluorouracilo/metabolismo , Metotrexato/farmacología , Animales , Médula Ósea/análisis , Médula Ósea/efectos de los fármacos , Cromatografía Líquida de Alta Presión , RatonesRESUMEN
Initial clinical trials have demonstrated that uridine (Urd) rescue given i.v. over at least 3 days can ameliorate 5-fluorouracil (FUra) toxicity; to avoid Urd-induced phlebitis in the peripheral veins of patients, a central vein is used. The latter necessity, along with the need for 3 days of i.v. administration, makes Urd rescue by parenteral means a cumbersome and complicated clinical procedure. It would appear preferable to use oral Urd; however, the oral Urd dose in the clinic is limited, as high doses cause diarrhea. Therefore, using a tumor-bearing murine model we investigated as to whether low doses of oral Urd coupled with a Urd phosphorylase inhibitor benzylacyclouridine (BAU), would effect safe rescue of FUra toxicity with preservation of antitumor activity. A high-dose FUra-containing drug combination that included parenteral Urd rescue was used as a control; other groups of tumor-bearing mice received the same drug combination, except that p.o. Urd was substituted for i.p. Urd. In the absence of BAU, p.o. Urd could effect rescue while maintaining an antitumor effect comparable to that obtained with i.p. Urd. When given concomitantly with BAU, a 50% reduction in the oral Urd dose (i.e., from 4,000 to 2,000 mg/kg) enabled the achievement of a comparable therapeutic index. Intraperitoneal Urd produces very high (6-8 mM) plasma and tissue Urd levels, which remain above 100 microM for at least 6 h. In contrast, neither oral Urd nor oral BAU alone raised plasma Urd concentrations above about 50 microM. However, the combination of oral Urd plus oral BAU gave a peak plasma Urd level of about 300 microM, and the level was maintained above 100 microM for 6 h. Following oral Urd administration, gut tissue levels of Urd were in the mM range and those of BAU were in the range of 10-20 micrograms/g tissue, a level sufficient to result in substantial inhibition of Urd phosphorylase. Oral Urd plus oral BAU appears to be a promising clinical alternative to parenteral administration of Urd for selective rescue of FUra toxicity.