Reversible suppression of mouse mammary tumor virus replication by prolonged glucocorticoid exposure.

Expression of mouse mammary tumor virus (MuMTV) in MJY-alpha mammary tumor cells was only transiently stimulated by exposure to 14 microM hydrocortisone (HC). Short-term HC treatment for 24-48 hours resulted in twofold-to-fivefold increases in levels of MuMTV polypeptide synthesis, MuMTV surface antigen expression, and MuMTV production. HC treatment also induced quantitative alterations in glycosylation of MuMTV precursors Pr79env and Pr76env. In contrast, prolonged exposure to 14 microM HC for 14-21 days decreased MuMTV polypeptide synthesis, surface antigen expression, and virion production to levels similar to untreated MJY-alpha cells. The pattern of MuMTV precursor glycosylation following prolonged HC treatment was identical to that detected in unexposed control cultures. Attenuation of glucocorticoid-mediated MuMTV stimulation was reversed either by exposure of treated cells to elevated (28 or 60 microM) concentrations of HC or by intermediate passage of treated cells in HC-free medium, suggesting additional levels of control of MuMTV replication.

Interference of mouse mammary tumor virus replication by PTT.119 [p-fluoro-L-phe-m-bis-(2-chloroethyl)amino-L-phe-met ethoxy HCl]--an antitumor agent.

PTT.119 [p-fluoro-L-Phe-m-bis-(2-chloroethyl)-amino-L-Phe-Met ethoxy HCl] is a new bifunctional alkylating compound that possesses both cytolytic and antiviral activities. Continuous treatment of mouse mammary tumor cells with 15 microM PTT.119 reduced production of the B-type retrovirus murine mammary tumor virus (MuMTV). MuMTV levels in PTT.119-treated cultures were reduced by 31% in the first 24 hours; an additional 24 hours of treatment resulted in a further reduction of 70%. These reductions were significantly greater than could be accounted for by the effects of PTT.119 tumor cell metabolism and viability. Under identical treatment conditions, equimolar concentrations of L-phenylalanine mustard reduced MuMTV production by only 3%. PTT.119 inhibition of MuMTV replication was also apparent when mammary tumor cells were exposed for periods as short as 0.5-4 hours; persistent decreases in virus production were detected even 7 days following tripeptide treatment. Analyses of the polypeptide composition of MuMTV by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that virions from these PTT.119-treated mammary tumor cultures contained significant reductions in the relative level of the nonglycosylated 24,000-dalton (p24) viral polypeptide associated with the nucleoprotein core. Decreases in p24 were observed in MuMTV produced in the presence of the tripeptide and 1-7 days after removal of PTT.119. Examination of the steady-state levels and rates of intracellular MuMTV protein synthesis suggested that PTT.119 interferes with late steps in MuMTV processing and maturation.

Characteristics of mammary tumor cultures from four mouse strains infected with mammary tumor virus.

The morphology and production of oncornaviruses in primary cell and explant mammary adenocarcinoma cultures derived from C3Hf, BALB/cNIV, BALB/cfC3H, and C3H mice were characterized. Cultures from the four mouse strains were morphologically similar; they were all epithelial and formed hemicysts and mounds. However, the numbers of cells containing mammary tumor virus (MTV) antigens and the production of cell-free MTV varied greatly among the established cultures. The percentages of MTV-positive cells in the cultures correlated with detection of cell-free MRV; immunodiffusion assays of virions from C3Hf and BALB/cNIV cultures were consistently negative for MTV antigens, whereas virions from BALB/cfC3H and C3H cultures were always positive. Synthesis of murine leukemia virus group-specific, soluble antigens was always observed in C3Hf, BALB/cNIV, and C3H cultures but only rarely observed in BALB/cfC3H cultures.

Effects of cytoskeletal disrupting agents on mouse mammary tumor virus replication.

Alterations in mouse mammary tumor virus (MMTV) production and composition were induced by exposure of mammary tumor cells to cytodisruptive agents. Treatment with 2.1 microM cytochalasin D (CD) for 24 h reduced MMTV yield by 80% and electron microscopic examination of these cells did not reveal budding virions. Immune precipitation and quantitative immunofluorescence studies demonstrated that CD had no significant effect on MMTV polypeptide synthesis or surface expression suggesting that CD inhibited late steps in MMTV maturation. Decreases in MMTV production were also observed as a result of 24 h exposure of the cells to 2.1 microM cytochalasin B (CB). However, an initial 70% increase in the levels of extracellular virions within the first 18 h of treatment preceded diminution of virus production. In addition, CB was unable to abrogate maturation and release of MMTV particles as revealed by electron microscopic evaluation of thin sections of treated cells. Colcemid at 0.28 microM had no effect on virus production during the first 24 h of exposure although MMTV yield was reduced by 60-70% after 36 h of treatment. Polypeptide profiles of MMTV purified from cell cultures treated with any of the three cytodisruptive agents were altered and included 5-7 polypeptides not typically present in MMTV from untreated cells. These cytodisruptive agents did not significantly affect viability and protein metabolism of MJY-alpha cells; the data suggest that alterations in MMTV replication were due to disruption of the cellular cytoskeleton.

PTT.119, p-F-Phe-m-bis-(2-chloroethyl)amino-L-Phe-Met ethoxy HCl, a new chemotherapeutic agent active against drug-resistant tumor cell lines.

PTT.119 [p-F-Phe-m-bis-(2-chloroethyl)amino-L-Phe-Met ethoxy HCl], a new synthetic tripeptide, was highly effective against the L-phenylalanine mustard (L-PAM) resistant (L1210/L-PAM and P388/L-PAM) tumor lines, as well as the sensitive L1210 leukemia. Cytolytic activity of PTT.119 against all three leukemias was significantly greater than equimolar doses of L-PAM. These in vitro results paralleled the significant increases in mean survival times of hosts and, in some cases, abrogations of tumor formation observed in the in vivo bioassays of PTT.119-treated L1210 and L1210/L-PAM cells. Dose-response studies failed to demonstrate cross-resistance to the tripeptide by L-PAM resistant cells. Doses of PTT.119 required to reduce the viable fraction by 50% (tissue culture dose 50, TCD50) or 100% (TCD100) were 1.3- to 3-fold lower for the L-PAM resistant cells than for the L1210 leukemia. In comparison, L-PAM was unable to completely eliminate cell survival; 0.2 to 3% of the cells in all three leukemias remained viable even at doses of 75 and 163 microM. In similar studies, L1210 leukemia cells made resistant to methotrexate (L1210 MTX) and cisplatin (L1210DDP) were also completely susceptible to PTT.119; TCD50 values of the two resistant lines were 1.94 microM for L1210 MTX and 0.525 microM for L1210DDP compared to 2.38 microM for the susceptible parent L1210S leukemia. Continuous low-dose PTT.119 treatment of MJY-alpha mammary tumor cells for 8 months and exposure of L1210 leukemia to escalating levels of tripeptide for over 100 passages failed to select or induce drug-resistant phenotypes in either cell line. PTT.119 appears to be a poor mutagen and is unlikely to readily increase the probability of drug-resistant mutants in the tumor cell populations.

Evaluation of p-F-Phe-m-bis-(2-chloroethyl)amino-L-Phe-Met-ethoxy HCl against transplantable and spontaneous murine neoplasia.

The therapeutic efficacy of PTT.119, p-F-Phe-m-bis-(2-chloroethyl)amino-L-Phe-Met-ethoxy HCl, was evaluated using the transplantable L1210 leukemia and Ridgway osteogenic sarcoma tumor lines and the spontaneous C3H/StRos mammary tumor and AKR leukemia tumor models. Given in a single i.p. dose at 5-10 mg/kg on day 2 or in two injections of 5-7 mg each on days 2 and 9 to BDf1 mice with peritoneal L1210 leukemia grafts, PTT.119 increased the life spans (ILS) of the population dying of tumor by 94%-313%. In addition, 10% of the mice receiving 7 mg PTT.119 on days 2 and 9 were free of L1210 leukemic grafts when autopsied at the end of the 70-day observation period. The average life span of AKR mice with Ridgway osteogenic sarcoma grafts was significantly increased from 36-40 days to greater than 79 days following one or two s.c. injections of 5, 7, or 12.5 mg/kg PTT.119. Administration of PTT.119 at 14 or 14 and 21 days after tumor graft not only induced regression of palpable tumors but resulted in the absence of grafts in 60%-70% of the mice in several of the treated groups on autopsy at 180 days. In contrast, spontaneous mammary tumors were less susceptible to PTT.119; an ILS of only 15%-38% was observed in C3H/StRos mice, which eventually succumbed to tumor. Nevertheless, the total regression of initial tumors and the absence of further tumor incidence (greater than 180 days) was confirmed by autopsy in 5%-10% of the C3H/StRos mice receiving multiple i.p. injections of 5 or 7.5 mg/kg PTT.119. The drug was highly effective against spontaneous AKR leukemia; multiple s.c. or i.p. injections for a total of 15-40 mg/kg PTT.119 increased the average 25-day life span up to 723% and sustained remission in 9%-40% of the animals for greater than 6 months.

Increased cancericidal activity of PTT.119; a new synthetic bis-(2-chloroethyl)amino-L-phenylalanine derivative with carrier amino acids. II. In vivo bioassay.

The cancericidal efficacy of a new synthetic tripeptide was demonstrated using both in vitro cultures and in vivo tumorigenic assays. The antitumor agent PTT.119 (p-F-Phe-m-bis-(2-chloroethyl)amino-Phe-Met ethoxy HCl) was highly effective against three virulent murine tumor models: the L1210 leukemia, MJY-alpha mammary tumor and B16 melanoma. Treatment of tumor cells for periods as short as 15 min to 4 h with concentrations of 1-50 micrograms PTT.119/ml irreversibly reduced tumor cell viability, as evidenced by vital dye exclusion and abrogation of tumor formation and prolongation of host survival. Examination of the sensitivity of mice to PTT.119 revealed that the in vitro antitumor activity of the synthetic tripeptide was exerted at concentrations easily attainable and well tolerated in vivo.

Increased cancericidal activity of PTT.119, a new synthetic bis-(2-chloroethyl)amino-L-phenylalanine derivative with carrier amino acids. I. In vitro cytotoxicity.

A new synthetic tripeptide (p-F-Phe-m-bis-(2-chloroethyl)amino-Phe-Met ethoxy HCl), PTT.119, was demonstrated to have significant cancericidal activity against seven in vitro tumor cell lines of different origins and etiologies and against primary human AMML, ALL, and hairy cell leukemias. Viabilities of each murine tumor and rabbit, marmoset, and human leukemia and lymphoma line were significantly reduced by treatment with 1-50 micrograms PTT.119 in media containing serum. Continuous 24-h exposure or pulse treatment as short as 15 and 30 min with the tripeptide resulted in irreversible damage to the tumor cells. Under identical treatment conditions, freshly isolated human leukemic cells, particularly ALL lymphoblasts, were even more susceptible to PTT.119 than any of the tested tumor cell models. Examination of the parameters of PTT.119 activity revealed that reductions of tumor cell survival were dependent on the concentration of the tripeptide. Prolongation of PTT.119 exposure from 15 min to 24 h increased the rates of tumor cell death but did not proportionally reduce the numbers of surviving cells. Assessment of tumor cell viabilities for 5 consecutive days following pulse exposure to PTT.119 demonstrated increasing reductions in tumor cell survival, which were greatest 5 days after treatment of PTT.119 was compared with its three parental components either as individual agents or as a mixture. Both the alkylator moiety, m-sarcolysin (m.L.SL) alone or together with p-fluoro-phenylalanine and L-methionine ethoxy HCl, and L-PAM (L-phenylalanine and L-methionine ethoxy HCl, and L-PAM (L-phenylalanine mustard), the p-isomer of m.L.SL, were 1,5- to 3-fold less cytotoxic to L1210 leukemia and MJY-alpha mammary tumor cells than PTT.119. Covalent linkage of the amino acid residues to m.L.SL yielded a molecule with greatly augmented cancericidal activity capable of acting against a broad spectrum of tumor cells.

Differential susceptibility and cytopathic alterations induced by PTT. 119, a new cancericidal compound, p-fluoro-Phe-L-Phe-m-bis-(2-chloroethyl)amino-L-Phe-Met ethoxy HCl.

PTT.119 is a new synthetic compound under assessment as a chemotherapeutic agent against neoplasia. Exposure of three diverse tumor cell lines, L1210 leukemia, MJY-alpha mammary tumor, and B16 melanoma to 1 to 50 micrograms PTT.119/ml for 15, 30 or 60 minutes significantly reduced cell survivals. Each tumor model was differentially susceptible to PTT.119 activity in the extent and kinetics of cell cytolysis. Pathological changes unique to each tumor cell type were also observed and included nuclear fragmentation and lobulation, formation of multinucleated cells, mitotic asynchrony and vacuolization of both nuclear and cytosol compartments. The data demonstrate the necessity of using more than one tumor system for the evaluation of compounds and suggest that PTT.119 exerted its cancericidal activity by more than one mechanism.

Increased yield of mouse mammary tumor virus (MMTV) by cultivation of monolayer-derived mammary tumor cells in suspension.

The MJY-alpha epithelial-like mammary tumor cell line was adapted for cultivation in suspension using a shaker culture technique. Replication of suspension (MJY-beta) cells was more sensitive than monolayer cells to decreases in the concentration of serum in the medium. Comparison of amino acid incorporation and lactate production rates revealed additional differences between monolayer and suspension cultures. In addition, growth in suspension resulted in 10- to 400-fold increases in mouse mammary tumor virus (MMTV) production by the mammary tumor cells. Increases in MMTV yield were detected within 48 h of culture initiation and MMTV production remained elevated throughout 20 cell passages in suspension. Exposure of MJY-beta cells to 14 microM hydrocortisone further increased MMTV yield two- to five-fold. The MJY-beta suspension cultures demonstrated that these epithelial-like cells do not require attachment to a solid substrate for replication or for MMTV production. Loss of structural polarization associated with growth as a monolayer resulted in stimulation of MMTV production greater than and independent of steroid exposure.

Modulation of mouse mammary tumor virus production in the MJY-alpha cell line.

Implantation of the mouse mammary tumor virus (MMTV)-producing mammary tumor cell line MJY-alpha into isogeneic mice elicited both humoral and T-cell responses against MMTV virion antigens. The carcinosarcomas which developed from the implanted cells showed a significant decrease in MMTV synthesis, compared with cells remaining in culture, which was detectable as early as 7 days after implantation and for five transplant generations. Electron microscopic examination of thin sections of the tumors revealed that intracytoplasmic A particles, budding particles, and cell-free MMTV B particles were all affected. However, immunofluorescence assays of tumor sections demonstrated the presence of MMTV viral antigens in the cells. Cell cultures initiated from first-, third-, and fourth-generation tumors were morphologically identical to the original in vitro cell line, although virus production was barely detectable. Analysis of the cultures by electron microscopy revealed a significant increase in MMTV virions after in vitro passage 3. Polypeptide profiles obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virions purified from these cultures were identical to MMTV. Immunodiffusion demonstrated the cross-reactivity between these virions and MMTV particles obtained from mouse milk. In vitro treatment of MJY-alpha cell cultures with rabbit anti-MMTV antiserum resulted in a reduction of extracellular MMTV virions, as well as alterations in their sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide patterns.

Alterations of a mouse mammary tumor virus glycoprotein with interferon treatment.

The effects of exogenous mouse interferon on the MJY-alpha mammary tumor cell line chronically infected with mouse mammary tumor virus (MMTV) were examined. Interferon at concentrations of 25 to 2,000 IU/ml in culture medium did not alter the growth rate or morphology of the cell layers. Electron microscopic examination of interferon-treated cells indicated a decrease in the numbers of A-type and budding B-type particles of MMTV. However, the levels of extracellular MMTV virions in the culture supernatants were not significantly reduced. Profiles of MMTV glycoproteins and nonglycosylated polypeptides obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virions purified from interferon-treated cultures revealed increases in the relative levels of the 60,000-dalton glycoprotein, gp60.

In vitro studies of 2,4-dihydroxyphenylalanine, a prodrug targeted against malignant melanoma cells.

We have evaluated the chemotherapeutic potential of 2,4-dihydroxyphenylalanine, a targeted prodrug that can be hydroxylated by tyrosinase (monophenol monooxygenase, EC 1.14.18.1) within melanoma cells to form the cellular toxin 2,4,5-trihydroxyphenylalanine (6-hydroxydopa). 2,4-Dihydroxyphenylalanine proved to be cytotoxic to both B-16 and Cloudman melanoma cells in vitro. The immediate effects of 2,4-dihydroxyphenylalanine included inhibition of DNA, RNA, and protein syntheses. In contrast, no decrease in macromolecular synthesis or viability was seen against cultures of MJY-alpha mammary tumor or L-1210 leukemia, two cell types that do not contain tyrosinase. Within the melanoma cultures, greater cytotoxicity was seen against melanotic (tyrosinase-containing) cells than against amelanotic (tyrosinase-lacking) cells. The cytotoxicity of 2,4-dihydroxyphenylalanine was blocked by 1-phenylthiourea, an inhibitor of tyrosinase. These results show that 2,4-dihydroxyphenylalanine is toxic to melanoma cells and that activation of 2,4-dihydroxyphenylalanine requires the presence of tyrosinase.

Tumor necrosis factor enhances murine SL3-3 retrovirus replication.

Synthesis of extracellular virions from NIH 3T3 cells productively infected with the murine leukemogenic SL3-3 retrovirus, was significantly increased by exposure to recombinant human tumor necrosis factor (rHuTNF). Compared to control cells SL3-3 virus production was 7-10-fold greater in cultures treated for 24 hours with 100-400 units of rHuTNF/cm2 of cell layer. In contrast, identical treatment of these cells with natural murine interferon alpha/beta, recombinant murine interferon-gamma, or recombinant human IL-2 consistently reduced SL3-3 virion production. Although both interferon (IFN) preparations decreased virion production by 25-95% within a 24 hour period, increased SL3-3 replication was observed following simultaneous addition of rHuTNF and either IFN. Increases in SL3-3 virus production did not require the constant presence of rHuTNF; elevated viral synthesis was detected 2 days after short pulse exposures of 30 minutes to 2 hours. Infected NIH 3T3 cells exposed to 200 units rHuTNF/cm2 of cell layer for 48 hours also continued to produce increased levels of SLS-3 virions 2 weeks after treatment.

Blocking of spleen cell activity against target mammary tumor cells by viral antigens.

Spleen cells from BALB/c females exposed to or neonatally infected with mammary tumor virus (MTV) are cytotoxic to MTV-induced mammary tumor cells in microcytotoxocity assay. This activity can be partially or completely blocked by pretreatment of spleen cells with MTV purified from milk. Murine leukemia virus (MuLV) has no effect. T cell responses of virgin and multiparous BALB/cfC3H females are effectively blocked. Non-T cell responses of multiparous BALB/cfC3H females or of virgin BALB/c females are blocked by some but not all of the MTV antigen preparations. MuLV, but not MTV, can block activity of spleen cells from MuLV-sensitized donors against target MuLV-producing tumor cells.

Mammary tumor and melanoma cell transport of PTT.119, a bis-(2-chloroethyl)amino-L-phenylalanine derivative with carrier amino acids.

Uptake of the bifunctional alkylating agent, PTT.119, p-F-Phe-m-bis-(2-chloroethyl)amino-L-Phe-Met-ethoxy-HCl, by the MJY-alpha mammary tumor and B16 melanoma cell lines occurred via two natural pathways for amino acid transport. The primary route of PTT.119 entry was the classical L System, whereas, the ASC System carrier was inconsistently operational for uptake of the tripeptide accounting for 5-6% of PTT.119 transport in the B16 melanoma cells. Tripeptide uptake by MJY-alpha mammary tumor cells did not occur via ASC carriers indicating that the mechanism of drug transport was dissimilar among the tumor cells. In spite of these differences the results support the hypothesis that amino acid residues covalently linked to an alkylating moiety do serve as carriers to enhance drug delivery.

Analytical and pharmacological studies on a new antineoplastic tripeptide, PTT.119.

The new synthetic tripeptide, p-fluoro-L-phenylalanyl-m-bis-(2-chloroethyl)-amino-L-phenylalanyl-methi onine ethylester hydrochloride, PTT.119, an alkylating agent, is currently undergoing preclinical trials as an antineoplastic agent. The molecular composition, C29H39N4O4SCl2F, was confirmed by desorption chemical ionization mass spectrometry with accurate mass measurement. A high-performance liquid chromatographic technique was developed for the quantification of PTT.119 in cell culture medium and serum. Incubation of 5 X 10(5) mammary tumor cells (MJY-alpha)/ml tissue culture medium with 25 micrograms PTT.119/ml for 60 min (37 degrees C) removed 68% of the tripeptide from the medium. This corresponds to an uptake of 51 fmol PTT.119/tumor cell. Cell death, assessed 5 days after treatment, was directly proportional to the time-dependent removal of PTT.119 from the cell culture medium.