Therapeutic effect reflected by plasma levels of a viral protein during combination chemotherapeutic treatment of mammary tumor-bearing mice.

Plasma concentrations of gp52, a Mr 52,000 glycoprotein of the mouse mammary tumor virus, have been measured during cyclophosphamide, doxorubicin (adriamycin) and 5-fluorouracil (CAF) treatment of mammary tumor-bearing CD8F1 mice. The value of plasma concentrations of gp52 as an indicator of CAF-mediated changes in tumor status was supported by each of the following findings: (a) CAF treatment did not interfere with the detection of elevated viral antigen levels in the plasma of tumor-bearing mice; (b) at 9-11 days after initiation of treatment, a significantly lower mean gp52 level, observed in the group of CAF-treated mice, provided definitive evidence of therapeutic effect; and (c) serial determinations of plasma gp52 levels in individual mice before, during, and after treatment provided a relative measure of therapeutic effect for each individual that was a reflection of corresponding changes in tumor size. Changes in viral antigen levels (i.e., decrease or increase) reflected inhibited tumor growth, as well as tumor regression. These findings demonstrate that plasma concentrations of gp52 can be utilized to provide an alternative measure of therapeutic effect in CAF-treated mice bearing significant tumor loads.

Properties of retrovirus-like particles produced by a human breast carcinoma cell line: immunological relationship with mouse mammary tumor virus proteins.

Clonal derivatives 8 and 11 of the T47D human breast carcinoma cell line release particles that have the biochemical characteristics of a retrovirus. Particles recovered from cultures of [3H]uridine-labeled clone 11 had a density of 1.18 g/ml and contained 60-70S and 35S RNAs associated with reverse transcriptase activity. The production of these particles was steroid-dependent. Clone 8 particles had a higher density, 1.195 g/ml, and their production was independent of steroid hormone. By RIA, antigens crossreactive with the 52,000-dalton envelope glycoprotein gp52, the major external protein of mouse mammary tumor virus, were found associated with these particles and in the media. Most of the gp52-related antigen was in soluble form, but it was enriched in the particle preparation. A lesser amount of antigen was distributed within the cultured cells. Absorption of rabbit antibody to gp52 with clone 11 particle preparations eliminated the ability of this antibody to detect immunocytochemically a crossreactive antigen previously localized in tissue sections of human breast carcinoma. These results indicate that the particle isolates from T47D contain the same gp52-related antigen found in human breast carcinomas and constitute an excellent source for the purification and characterization of this antigen.

Incorporation of 5-fluorouracil into murine bone marrow DNA in vivo.

Although 5-fluorouracil (FUra) is readily incorporated into RNA, the possibility of its being incorporated into DNA in substantial amounts has only recently been recognized. Examination of nucleic acids prepared from tumor-bearing BALB/c X DBA/8 F1 mice labeled with [3H]FUra in vivo revealed very little alkali-stable, acid-precipitable radioactivity in tumor and only small amounts in intestine. However, substantial amounts were detected in bone marrow. Pretreatment of mice with low-dose thymidine (500 mg/kg) increased the incorporation of FUra into RNA but did not change the amount incorporated into alkali-stable material. The net result was a reduction in the fraction of the total in an alkali-stable form. Formation of DNA containing FUra residues is substantially reduced if the mice receive very high doses of thymidine along with the labeled FUra, presumably through competition from an expanded deoxythymidine triphosphate pool. Bone marrow nucleic acids labeled with 32P and [3H]FUra were analyzed by cesium sulfate gradients. Two distinct peaks of tritium radioactivity were observed that band with 32P radioactivity at the densities of RNA and DNA. Pretreatment with alkali destroyed the (32P/3H)RNA peak, but not the DNA peak. Cesium sulfate-purified DNA containing FUra residues was digested with pancreatic DNase and venom phosphodiesterase. The resulting nucleotides were analyzed by high-pressure liquid chromatography. The majority of the radioactivity cochromatographed with 5-fluorodeoxyuridine monophosphate marker. No radioactivity was detected in the regions corresponding to fluorouridine monophosphate or deoxyuridine monophosphate, although radioactivity was detected cochromatographing with deoxythymidine monophosphate. After digestion with alkaline phosphatase, the majority of the radioactivity cochromatographed with fluorodeoxyuridine (and some thymidine). These results confirm previous observations of FUra incorporation into DNA of tissue culture cells.

Increased incidence of mouse mammary tumor virus-related antigen in Tunisian patients with breast cancer.

Biopsies obtained from 74 Tunisian women with breast cancer (33 cases), benign breast disease (17 cases), and cervical cancer (24 cases) were assayed for the presence of an antigen cross-reacting with gp52 of the mouse mammary tumor virus (MMTV) in order to determine the frequency and possible prognostic significance of this antigen in a form of rapidly progressing breast cancer designated poussée évolutive or PEV. Antigen was detected in 23/33 breast carcinomas (70%) but in none of the 41 control specimens. An evaluation of reactivity according to tumor aggressiveness and survival could be performed in retrospect on 29 of the breast cancer patients with a follow-up of up to 11 years. The frequency of gp52-related antigen was similar in the patients with the most aggressive form of PEV with inflammatory signs (8/12 or 67% positive) and those breast cancer patients without PEV (12/17 or 71% positive). Within each of the two groups, PEV+ and PEV 0, no correlation was observed between the presence or absence of antigen and the disease-free interval or survival. We conclude that the identification of gp52-related antigens in the breast cancer biopsies from North African women has implications different from those observed in other populations. While thus far not indicative of disease aggressiveness and prognosis, the higher frequency of detectable antigen in comparison to biopsies obtained from patients born in the United States and Europe may have relevance to the etiology and pathogenesis of the disease.

Effect of Uridine on the Metabolism of 5-Fluorouracil in the CD8F 1 Murine Mammary Carcinoma System.

The effect of uridine on the incorporation of 5-fluorouracil into RNA and the inhibition of DNA synthesis by the FdUMP block of thymidylate synthetase was studied in the CD8F1 murine mammary carcinoma system. The administration of exogenous uridine resulted in about a one third reduction of 5-fluorouracil in RNA of tumor and normal tissues. However, unlike thymidine, uridine was unable to reverse the early, partial inhibition of DNA synthesis. The amount of fluorouridine nucleotides and (5-fluorouracil)RNA formed in various tissues correlates with the level of orotate phosphoribosyl transferase activity suggesting that the major pathway for activation of 5-fluorouracil to nucleotide form in these tissues is via phosphoribosyl transferase. Enzyme preparations from three different murine tumors convert about 15 times as much 5-fluorouracil to FUMP as they do uracil to UMP. In contrast, the ratio of FUMP to UMP formed in enzyme preparations from gut and bone marrow is lower, 2-6 fold. However, in none of these tissues was the in vitro conversion of 5-fluorouracil to FUMP or incorporation into RNA substantially inhibited by uracil. Examination of tumor, gut and bone marrow uridine nucleotide pools showed that the thymidine-uridine-5-fluorouracil schedule does increase uridine nucleotide pools. Thus, the reduction in 5-fluorouracil in RNA is probably not due to inhibition of the conversion of 5-fluorouracil to FUMP by uracil (derived from phosphorylase cleavage of uridine) but, rather, is probably due to the elevated levels of UTP. We conclude that the protection from 5-fluorouracil toxicity afforded by the addition of uridine is due to the reduction in 5-fluorouracil in RNA rather than by reversal of the FdUMP block on thymidylate synthetase.

Improved therapeutic index with sequential N-phosphonacetyl-L-aspartate plus high-dose methotrexate plus high-dose 5-fluorouracil and appropriate rescue.

Although clinical trials of high-dose methotrexate (MTX) sequenced before 5-fluorouracil (FUra) with leucovorin (LV) rescue apparently have resulted in increased numbers of tumor responses, this increased antitumor activity often has been accompanied with toxicity. The present report describes an attempt to improve therapeutic results with this drug combination by appropriate metabolic modulation in the preclinical BALB/c X DBA/8 F1 murine breast tumor model. A LV rescue schedule consisting of 300 mg/kg administered at 4.5 and 19.5 hr after high-dose MTX (300 mg/kg/week for 3 weeks) prevented MTX toxicity. When FUra was administered 2.5 hr after MTX (with LV rescue), the dose of FUra had to be decreased, and we could not obtain convincing evidence for a differential cytotoxic effect on tumor versus normal host tissue. However, when a delayed uridine rescue schedule was added to protect the host from the toxic activity of FUra, the FUra dose could be increased even in the presence of high-dose MTX, and the therapeutic result was enhanced significantly without an increase in host toxicity. Finally, it was possible to add N-phosphonacetyl-L-aspartate to this drug combination (in the appropriate sequence: N-phosphonacetyl-L-aspartate before high-dose MTX-before high-dose FUra, followed by double rescue with LV and uridine) without producing increased toxicity to yield a significant increase in partial tumor regression rate. The biochemical rationale for the selection and sequence of administration of these agents is discussed.

Therapeutic utility of utilizing low doses of N-(phosphonacetyl)-L-aspartic acid in combination with 5-fluorouracil: a murine study with clinical relevance.

Doses of N-(phosphonacetyl)-L-aspartic acid (PALA) lower than those required for therapeutic activity against the spontaneous murine breast tumor (BALB/c X DBA/8 F1) were found to produce significant depression in uridine triphosphate pools in the tumor. Using such low, nontherapeutic, but biochemically active doses of PALA in combination with 5-fluorouracil (FUra(, it was possible to maintain the dose of FUra at its full maximum tolerated single agent dose. In comparison with the maximum tolerated dose of FUra alone, the combination of FUra plus low-dose PALA produced a significant increase in the level of tumor FUra-containing RNA, only a slight increase in intestinal FUra-containing RNA, and no increase in bone marrow FUra-containing RNA. These biochemical results correlated with the therapeutic findings of significantly increased antitumor activity without an increase in host toxicity. A review of currently reported PALA plus FUra clinical protocols reveals that the experimental parameters which have produced successful therapeutic results in the laboratory (i.e., a low-PALA:high-FUra dosage ratio) have not yet been translated into clinical trial. Final judgment on the clinical efficacy of PALA:FUra combinations must await the results of proposed low-PALA:high-FUra clinical trials.

Modulation of 5-fluorouracil-induced toxicity in mice with interferon or with the interferon inducer, polyinosinic-polycytidylic acid.

Partially purified preparations of mouse interferon, administered during the 2-day period following the administration of a toxic dose of 5-fluorouracil (FUra), yielded significant protection from mortality in BALB/c X DBA/2 F1 mice. Protection against FUra-induced toxicity was also observed when the interferon inducer polyinosinic-polycytidylic acid (poly I X poly C) was administered with FUra. The temporal relationship between the administration of poly I X poly C and FUra was found to be a critical determinant of the intensity of toxic manifestations. In relation to FUra alone, poly I X poly C could enhance (when administered 48 hr before FUra), diminish (when administered together with FUra), or not affect (when administered 48 hr after FUra) the degree of resultant toxicity. Cytofluorometric analysis of the DNA content of bone marrow cells indicated a transient period (about 42 hr) of inhibition of cell cycling following the administration of poly I X poly C, followed by reentry into cycle (between 42 and 66 hr) and a return to normal cycle phase distribution by 90 hr. This disturbance of the kinetic pattern of cell cycling in bone marrow would explain the administration time-dependent variability of the effect of poly I X poly C on FUra toxicity, since FUra is known to be a cell cycle-specific cytotoxic drug. Potential practical application of this observation to the clinical use of FUra in cancer therapy is discussed.

Tumor-associated antigens produced by mouse mammary tumor cells in artificial capillary culture.

Mouse mammary tumor cells were cultured on a three-dimensional bundle of semipermeable hollow fibers as a prototype system for large-scale in vitro production of mammary tumor-associated antigens. Cells were seeded onto the surface of the hollow fibers that served as an artificial capillary network. The cells grew to tissue-like densities within 3 weeks, achieving the high density and three-dimensional intercellular relationships known to potentiate expression of murine mammary tumor virus (MuMTV) antigens. Significant levels of a 52,000-relative-molecular-weight (Mr) glycoprotein antigen of MuMTV (gp52) were detected on the cell side of the semipermeable membrane of the hollow fibers in a yield at least fiftyfold greater than that produced by monolayer cultures. Antigen could be collected from capillary cultures for 6-12 weeks, rather than days from monolayer cultures. Further, gp52 was collected in an undegraded state. Perfusates contained lesser amounts of cross-reactive antigenic species that were predominantly smaller than 50,000 Mr, which suggests that the fibers may be used to separate antigens from their degradation products. The production of nonviral, mammary tumor-associated antigens by cells on artificial capillaries was also demonstrated. Artificial capillary cell culture systems provide a means to obtain significant quantities of tumor-relevant antigens in a semipurified natural state.

High-dose 5-fluorouracil with delayed uridine "rescue" in mice.

Because of the association between the incorporation of 5-fluorouracil (FUra) into RNA and cytotoxicity, uridine was examined for potential selective reduction of host toxicity. Male BALB/c x DBA/2 F1 mice (tumor free or bearing advanced colon tumor 26) were used. Two uridine schedules (each beginning 2 hr after FUra) have been successful: (a) uridine at 800 mg/kg every 2 hr for three doses followed 18 hr later by uridine at 800 mg/kg every 2 hr for four doses; or (b) two doses of uridine at 3500 mg/kg separated by an 18-hr interval. With two doses of uridine at 3500 mg/kg, the 50% lethal dose for a single dose of FUra in tumor-free mice was increased 68% from 190 to 320 mg/kg. White blood cell levels were depressed 67% after a single dose of FUra at 200 mg/kg, whereas white blood cells were depressed by only 39% when the same dose of FUra was followed by uridine rescue. In tumor-bearing mice, uridine rescue reduced FUra-induced host toxicity without significant loss of antitumor activity. Even more striking results were obtained with a combination containing N-(phosphonacetyl)-L-aspartate (200 mg/kg) and 6-methylmercaptopurine riboside (25 mg/kg), administered 24 hr before FUra. In this drug combination, the maximum tolerated dose of FUra is 40 mg/kg on a weekly schedule. With uridine rescue, FUra can be doubled to 80 mg/kg without increasing toxicity, resulting in significantly improved antitumor activity. Examination of the effect of uridine rescue on the incorporation of FUra into RNA and the subsequent recovery from inhibition of DNA synthesis in bone marrow versus tumor revealed that the uridine rescue schedule resulted in relatively faster clearance of FUra from RNA of both tissues but a striking enhancement of the rate of recovery of DNA synthesis only in the bone marrow.

Diagnosis of primary breast carcinoma through immunohistochemical detection of antigen related to mouse mammary tumor virus in metastatic lesions: a report of two cases.

Recent investigations have established that approximately half of human breast carcinomas contain an immunohistochemically detectable antigen which is cross-reactive with the 52000-dalton major glycoprotein (gp52) of the mouse mammary tumor virus (MMTV). This antigen can be localized in paraffin-embedded sections of routinely fixed tissues using heterologous antibodies to gp52 or MMTV. This report describes two patients with metastatic carcinoma in axillary lymph nodes without any clinical evidence of a primary lesion in the breast or elsewhere. The localization of the gp52-related antigen in paraffin-embedded sections of both metastatic lesions suggested the presence of primary mammary carcinoma. In both instances, this suggestion was ultimately confirmed by the finding of primary lesions in which the gp52-related antigen was also found.

Improving the anti-tumor activity of 5-fluorouracil by increasing its incorporation into RNA via metabolic modulation.

The experiments described here illustrate the use of metabolic modulation to improve the therapeutic effectiveness of 5-fluorouracil (5FUra) in two murine tumor systems (CD8F1 mannary carcinoma and CD2F1 colon tumor 26). The manipulations chosen were based on the assumption that a major fraction of the anti-tumor activity of 5FUra is due to its incorporation into RNA and that the resulting 5FUra-RNA creates difficulty for a variety of cellular mechanisms requiring RNA processing and function. This hypothesis leads to the prediction that thymidine would promote the anti-neoplastic effect of 5FUra due to the following possible interactions: (i) sparing 5FUra from catabolic degradation by saturating the relevant enzymes wit thymidine; (ii) selective arrest of normal cells due to feedback inhibition of robonucleotide reductase by the accumulating thymidine triphosphate (TTP); and (iii) the high levels of TTP would also be expected to repress the anabolic conversion of 5FUra to the deoxy derivatives, thus preserving it for entry into RNA. The data show that thymidine (and certain other nucleosides) does in fact markedly stimulate the incorporation of 5FUra into nuclear RNA and that this event is paralleled by a striking icrease in anti-tumor activity. Kinetic analysis reveals that, although the injection of 5FUra leads to an immediate cessation of thymidylate synthetase activity, DNA synthesis continues at a lower rate for 12 hr and then ceases completely. At this point, in contrast to the earlier partial inhibition, the addition of thymidine fails to restore the ability of the tumor cells to synthesize DNA.

Protection by testosterone from fluorouracil-induced toxicity without loss of anticancer acitvity against autochthonous murine breast tumors.

The present paper describes the specific amelioration of 5-flourouracil (FUra)-induced host toxicity (manifest in body weight loss, leukopenia, and mortality) by testosterone in the spontaneous, autochthonous CD8F1 (BALB/c x DBA/8F1) murine breast tumor model. Administration of testosterone did not affect the growth rate of these hormone-independent tumors, and, most importantly the antitumor activity of FUra was not reduced in testosterone-treated mice. Therefore, the net result of treatment with the combination of FUra and testosterone was an increase in the selective antitumor specificity of FUra.

RNA primer used in synthesis of anticomplementary DNA by reverse transcriptase of avian myeloblastosis virus.

When either the homologous RNA (avian myeloblastosis virus RNA) or a heterologous RNA (poliovirus RNA) was used as a template, the anticomplementary DNA synthesized in vitro by avian myeloblastosis virus reverse transcriptase (RNA-directed DNA nucleotidyltransferase, EC 2.7.7.7) was primed by fragments of the original RNA template that usually had adenosine at their 3' ends. When we used phage T/ RNA ligase (EC 6.5.1.3) to label the 3' end of the RNA template fragments contained in the RNA . cDNA hybrid intermediate, adenosine was found to be the principal nucleoside carrying the label. We infer from these results that the ribonuclease H (hybrid nuclease) activity of the reverse transcriptase creates fragments of the original RNA template with adenosine as the principal 3' terminus and that these fragments serve as primers for the synthesis of anticomplementary DNA.

An overview of thymidine.

This review summarizes a body of information suggesting that proper metabolic modulation with certain metabolites can sensitize tumor cells to anti-metabolites, and others can de-sensitize (i.e. protect) normal cells from the toxicity of anti-metabolites. This new approach offers the possibility of increasing the selectivity of drug therapy, with the promise of a real advance in cancer chemotherapy. The metabolite thymidine (TdR), long used as a cell synchronizing agent, is known to exert this effect in vitro by metabolic modulation of a number of enzymes in the salvage pathway to DNA synthesis. Against this biochemical background, in vivo effects of TdR employed as an agent for cancer therapy are reviewed as follows: 1) TdR alone, and in combination with, 2) Methotrexate (MTX), or 3) 5-Fluorouracil (FU), or 4) Cytosine arabinoside (ara-C). TdR is shown in all instances either to protect against host toxicity (eg. MTX), or to potentiate the anti-tumor effect (eg. FU and ara-C). Findings are also presented that a sequential schedule of MTX prior to TdR prior to FU is important for the optimal therapeutic activity of these drugs. The biochemical basis for the MTX leads to FU augmentation is reportedly due to increased activation of FU by MTX (acting indirectely). On the basis of this biochemical insight, a completely different chemotherapeutic agent methyl-mercaptopurine raboside (MMPR) was substituted for MTX, resulting in a dramatic potentiation of anticancer activity. Metabolic modulation with still other metabolites (UR) and a hormone (testosterone) was demonstrated to protect from host toxicity due to certain anti-cancer agents without offsetting anti-tumor activity. The ability to prevent leukopenia by these means was particularly impressive. Clinical trials have been initiated with TdR alone, TdR + MTX, and TdR + FU; the available clinical data are summarized.