Long-term selection for litter size in swine results in shifts in allelic frequency in regions involved in reproductive processes.

High-density genotype data were analyzed in three lines of swine that express substantial variation in sow fertility to uncover regions of the genome potentially influenced during selection for litter size traits. The experimental lines examined include the Nebraska Index Line (NIL), which has been subjected to long-term selection for litter size; a control line derived from the same population that founded NIL; and a commercial Duroc × Hampshire (D × H) population, in which no selection for litter size was practiced. Regions of the genome potentially affected by selection for litter size traits in NIL were determined by multiple lines of evidence, including altered allelic frequency compared to the other lines, loss of heterozygosity and relative extended haplotype homozygosity. Additionally, a genome-wide association study for litter size traits was conducted in a population based on NIL and commercial maternal line genetics. Several genomic regions identified as putative signatures of selection overlapped with QTL for litter size traits. One of these regions, located on SSC2 (13-14 Mb), includes the candidate gene P2X3R, which plays a role in implantation and sustained release of hormones associated with reproductive processes. Sequencing identified synonymous SNPs in P2X3R that are fixed in NIL but polymorphic with nearly equal frequencies in the D × H line, indicating a potential role of P2X3R in sow fertility. These results suggest that data derived from these lines can help to uncover and understand a portion of the genetic variance associated with fertility traits in swine.

Genomic analysis of the differential response to experimental infection with porcine circovirus 2b.

Porcine circovirus type 2 (PCV2) is the etiological agent of a group of associated diseases (PCVAD) that affect production efficiency and can lead to mortality. Using different crossbred lines of pigs, we analyzed host genetic variation of viral load, immune response and weight change following experimental infection with a PCV2b strain (n = 386). Pigs expressed variation in the magnitude and initiation of viremia and immune response recorded weekly until 28 days post-infection. A higher viral load was correlated with weight gain (r = -0.26, P < 0.0001) and presence of PCV2-specific antibodies (IgM, r = 0.26-0.34, P < 0.0001; IgG, r = 0.17-0.20, P < 0.01). In genome-wide association analyses of the responses at different time points, the proportions of phenotypic variation explained by combined effects of 56 433 SNPs were 34.8-59.4% for viremia, 10.1-59.5% for antibody response and 5.6-14.9% for weight change. Relationships between genomic prediction of overall viral load and weight gain during the first weeks of challenge were negative (-0.21 and -0.24 respectively, P < 0.0001). Individuals that carried more favorable alleles across three SNPs on SSC9 (0.60 Mb) and SSC12 (6.8 and 18.2 Mb) partially explained this relationship, having lower viral load (P < 0.0001); lower viremia at day 14 (P < 0.0001), day 21 (P < 0.01) and day 28 (P < 0.05) and greater overall average daily gain during infection (ADGi ; P < 0.01), ADGi at week 3 (P < 0.001) and week 4 (P < 0.01). These additive genetic relationships could lead to molecular solutions to improve animal health and reduce production costs.

Genome-wide prediction of age at puberty and reproductive longevity in sows.

Traditional selection for sow reproductive longevity is ineffective due to low heritability and late expression of the trait. Incorporation of DNA markers into selection programs is potentially a more practical approach for improving sow lifetime productivity. Using a resource population of crossbred gilts, we explored pleiotropic sources of variation that influence age at puberty and reproductive longevity. Of the traits recorded before breeding, only age at puberty significantly affected the probability that females would produce a first parity litter. The genetic variance explained by 1-Mb windows of the sow genome, compared across traits, uncovered regions that influence both age at puberty and lifetime number of parities. Allelic variants of SNPs located on SSC5 (27-28 Mb), SSC8 (36-37 Mb) and SSC12 (1.2-2 Mb) exhibited additive effects and were associated with both early expression of puberty and a greater than average number of lifetime parities. Combined analysis of these SNPs showed that an increase in the number of favorable alleles had positive impact on reproductive longevity, increasing number of parities by up to 1.36. The region located on SSC5 harbors non-synonymous alleles in the arginine vasopressin receptor 1A (AVPR1A) gene, a G-protein-coupled receptor associated with social and reproductive behaviors in voles and humans and a candidate for the observed effects. This region is characterized by high levels of linkage disequilibrium in different lines and could be exploited in marker-assisted selection programs across populations to increase sow reproductive longevity.

A small, nonpeptidyl mimic of granulocyte-colony-stimulating factor [see commetns].

A nonpeptidyl small molecule SB 247464, capable of activating granulocyte-colony-stimulating factor (G-CSF) signal transduction pathways, was identified in a high-throughput assay in cultured cells. Like G-CSF, SB 247464 induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro. It also elevated peripheral blood neutrophil counts in mice. The extracellular domain of the murine G-CSF receptor was required for the activity of SB 247464, suggesting that the compound acts by oligomerizing receptor chains. The results indicate that a small molecule can activate a receptor that normally binds a relatively large protein ligand.

Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

A general review of competition genetic effects with an emphasis on swine breeding.

A review of previous studies is presented on estimates of genetic parameters and responses to selection with traditional breeding approaches, on correlations between agonistic behavior and growth performance, and on theoretical frameworks for selection incorporating interactions among individuals and on practical methods for incorporating competition effects in breeding programs.

Biochemical parameters of resistance of an adriamycin-resistant subline of P388 leukemia to emetine, an inhibitor of protein synthesis.

The effects of emetine on protein and DNA synthesis in vitro and in vivo were compared in P388 leukemia cells (P388/S) and in an adriamycin-resistant subline of P388 leukemia (P388/ADR), which was completely cross-resistant in vivo to emetine. In P388/ADR cells in vitro no apparent resistance to emetine was found; no difference in cytotoxicity was evident in P388/S or P388/ADR cells exposed to emetine in vitro for 1 or 6 hours. Protein and DNA synthesis was inhibited to a similar extent in P388/S and P388/ADR cells at equivalent concentrations of the drug. However, inhibition of protein synthesis by emetine in P388/ADR cells was more reversible than in P388/S cells when the cells were exposed to emetine and subsequently incubated in drug-free medium for 1 hour prior to addition of labeled L-leucine. Differences between P388/S and P388/ADR cells were evident in vivo. The duration of inhibition (greater than 90%) of protein and DNA synthesis in P388/ADR cells was about 8 hours compared to 24 hours in P388/S cells following administration of a therapeutic dose of 25 mg emetine/kg to tumor-bearing mice. The level of radioactivity in the P388/ADR cells 24 hours after in vivo administration of the emetine analog, (+/-)-[3'-14C]2,3-dehydroemetine, was only 26% of that in P388/S cells. This evidence suggests that the resistance of P388/ADR to emetine is due to decreased retention of the drug.

A facile method of purification of neocarzinostatin, an antitumor protein.

A three-step column chromatographic method for the purification of neocarzinostatin (NCS) from a crude preparation was described. The purified material was homogeneous by acrylamide gel electrophoresis, isoelectric focusing, amino terminal analysis, and immunologic criteria. Purified NCS was 40 times as active in the inhibition of growth of Sarcina lutea and twice as active against CCRF-CEM human leukemia cells in vitro as was the starting material. When assayed against P388 and L1210 mouse leukemias in vivo, the purified material showed a median increase in life-span of 119 and 72%, respectively.

Nonproductive rearrangement of DNA topoisomerase I and II genes: correlation with resistance to topoisomerase inhibitors.

Topoisomerase inhibitors comprise an important group of agents that is used in cancer treatment. Because the development of resistance to cancer chemotherapeutic agents represents a major limitation of cancer chemotherapy, we investigated the mechanism of resistance by murine P388 leukemia to camptothecin (topoisomerase I inhibitor) or amsacrine (topoisomerase II inhibitor). The resistant cells contained reduced levels of topoisomerase activity and messenger RNA. The topoisomerase gene of these cells was rearranged (only in one allele) and hypermethylated. These topoisomerase gene alterations probably resulted in reduced transcription and, thus, enzyme production, which was correlated with resistance to the topoisomerase inhibitor.

Activity of topotecan, a new topoisomerase I inhibitor, against human tumor colony-forming units in vitro.

BACKGROUND: Topotecan [(S)-9-dimethylaminomethyl(10-hydroxy-camptothecin), NSC 609699, SK&F 104864A], a semisynthetic analogue of the natural product camptothecin, is a cell cycle-specific drug that exerts antineoplastic activity through inhibition of topoisomerase I. Currently, topotecan is undergoing phase I and early phase II clinical trials. The dose-limiting toxicity for topotecan is myelosuppression. PURPOSE: Our purpose was to determine plasma concentrations and exposure times necessary for optimal clinical activity and tumor types that may be responsive in phase II clinical studies of topotecan. METHODS: A soft-agar cloning system assay was used to determine the in vitro effects of topotecan against cells from biopsy specimens of colorectal, breast, lung, ovarian, renal cell, and gastric cancers and cancers of unknown primary origin. We studied 141 freshly explanted tumor specimens, using 1-hour exposure to topotecan, and 80 were studied using continuous exposure. A decrease in tumor colony formation resulting from drug exposure was considered an in vitro response if survival of colonies was up to 50% of that in controls. RESULTS: With 1-hour exposure, in vitro responses were seen in 10% and 25% of assessable tumor specimens at final topotecan concentrations of 1.0 and 10.0 micrograms/mL, respectively. With continuous exposures at concentrations of 0.1 and 1.0 micrograms/mL, in vitro response rates were 34% and 76%, respectively. Specific activity was seen against colorectal, breast, non-small-cell lung, ovarian, and renal cell cancers, with responses observed in 27%, 25%, 32%, 39%, and 83%, respectively, of assessable tumor specimens after continuous exposure to 0.1 micrograms/mL topotecan. A subset of tumor specimens resistant to doxorubicin or fluorouracil was sensitive to topotecan, and the difference in sensitivity was statistically significant. In addition, some of the tumor specimens resistant to cyclophosphamide and etoposide were also sensitive to topotecan. CONCLUSIONS: Topotecan appears to be active in vitro against a variety of human tumors, including a subgroup resistant in vitro to standard antineoplastic agents. If plasma levels of 0.1 micrograms/mL can be achieved for prolonged periods of time in ongoing clinical trials, topotecan should have substantial clinical activity. IMPLICATIONS: Further clinical development of topotecan is warranted.

Effects of local tumor hyperthermia on the growth of solid mouse tumors.

The sensitivity to local tumor hyperthermia (43 degrees, 60 min) of a spectrum of eight different solid mouse tumors (Lewis lung carcinoma, M5076 ovarian carcinoma, colon carcinoma 38, colon carcinoma 26, mammary adenocarcinoma C3HBA, mammary adenocarcinoma 16C, glioma 26, and B16 melanoma) was investigated. A microwave (2.45-GHz) apparatus produced localized heating of the tumors without generation of whole-body hyperthermia. The temperature at the center of the heated tumors was regulated to within +/- 0.1 degrees while the temperature uniformity within the tumor was +/- 0.5 degrees. The local hyperthermia treatments reduced the size and retarded the growth of the treated tumors compared with control values for each of the tumors tested. The faster-growing Lewis lung carcinoma and B16 melanoma were the least responsive to treatment, while the slower-growing colon 38 and M5076 ovarian carcinomas were the most responsive. Multiple treatments resulted in longer grwoth delays and greater tumor growth inhibition than did single treatments. No consistent difference in life span between the control and treated groups was measured, and only five of 188 treated animals were cured.

Decreased retention of actinomycin D as the basis for cross-resistance in anthracycline-resistant sublines of P388 leukemia.

Sublines of P388 leukemia resistant to Adriamycin and daunorubicin were cross-resistant to actinomycin D in vivo and in vitro. The Adriamycin-resistant cell line was 1000-fold resistant to actinomycin D on 1-hr exposure in vitro and 370-fold resistant when exposed to the drug for 16 hr. The immediate binding of radioactive actinomycin D to sensitive and resistant cells was similar, and the uptake of the drug by the resistant cells was only about 27% less than the rate of uptake by sensitive cells. There was a dramatic difference in efflux of drug from sensitive and resistant sublines. Equivalent cytotoxicity of actinomycin D for the sensitive and resistant sublines was obtained at concentrations of the drug that resulted in approximately equivalent levels of net retention of actinomycin D (retained drug minus background levels of immediate binding of the drug to the cells). Incubation of cells in the presence of actinomycin D plus either Tween 80 or acridine orange incresed the rate of uptake and the percentage of actinomycin D retained by the resistant cells on short-term assays but did not reverse the resistance. It is concluded that these tumors must retain appreciable concentrations of actinomycin D for several hr in order to be killed. The anthracycline-resistant sublines are cross-resistant to actinomycin D by virtue of their inability to retain the drug.

Collateral sensitivity to N-(phosphonacetyl)-L-aspartic acid in a line of P388 leukemia cells selected for resistance to L-(alpha S, 5S)-alpha-amino-3- chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin).

Administration of N-(phosphonacetyl)-L-aspartic acid (PALA) is ineffective in treating mice bearing the parent P388 leukemia line; however, such treatment becomes highly effective when a cell line, P388/ACIA, derived from P388/0 was selected for resistance to another antimetabolite, acivicin. The observed phenomenon of collateral sensitivity is associated with a significantly higher inhibition of the specific activity of carbamyl phosphate synthetase II, pyrimidine nucleoside kinases, adenine phosphoribosyl transferase, and hypoxanthine phosphoribosyl transferase in the PALA-sensitive line, P388/ACIA. Twenty-four hr following administration of PALA, 200 mg/kg, the 10% lethal dose i.p. to tumor-bearing mice, the intracellular concentrations of uridine triphosphate and cytidine triphosphate were decreased in the P388/ACIA, PALA-sensitive cells, whereas no significant change in the corresponding nucleotide pool sizes was observed in P388/0, PALA-resistant line. Moreover, the purine nucleotide pool demonstrated a significant expansion of adenosine triphosphate and guanosine triphosphate only in the P388/ACIA line following a similar treatment with PALA. It is proposed that the imbalance in the generation of pyrimidine and purine nucleoside triphosphate pools may explain the observed collateral sensitivity to PALA in P388/ACIA leukemia line.

Active efflux of daunorubicin and adriamycin in sensitive and resistant sublines of P388 leukemia.

Significantly decreased uptake and retention of daunorubicin (DAU) and Adriamycin (ADR) have been reported in sublines of P388 leukemia resistant to these anthracyclines. We studied the effects of inhibitors of oxidative phosphorylation on uptake and retention of DAU and ADR in order to characterize the transport process for these anthracyclines and to clarify further the alteration in resistant cells. In glucose-free medium, uptake of DAU and ADR was accelerated by metabolic inhibitors to a greater extent in resistant cells than in sensitive cells. Under these conditions, drug uptake was similar in sensitive and resistant cells. When glucose was added to the incubation medium in the presence of 2,4-dinitrophenol, efflux of DAU from both sensitive and resistant cells was observed. Net efflux of ADR was also observed with the resistant cells, whereas glucose markedly inhibited 2,4-dinitrophenol-stimulated uptake of ADR by the sensitive cells. Furthermore, in sensitive and resistant cells preloaded with ADR and DAU, efflux of the drugs was inhibited by the addition of 2,4-dinitrophenol. These results suggest that there is an active outward transport mechanism for anthracyclines in P388 leukemia cells and that enhanced activity of this efflux process renders cells highly resistant to the cytostatic and cytotoxic effects of ADR and DAU.

Antitumor activity of N-(phosphonacetyl)-L-aspartic acid, a transition-state inhibitor of aspartate transcarbamylase.

N-(Phosphonacetyl)-L-aspartate (PALA) is an analog of the transition state for the aspartate transcarbamylase reaction and has been reported previously to be a potent and specific inhibitor of de novo pyrimidine nucleotide biosynthesis. It is now shown that PALA has considerable antitumor activity against certain transplantable tumors in mice. PALA, unlike other antimetabolites, was less effective against ascitic leukemias than against two solid tumors, B16 melanoma and Lewis lung carcinoma. Another solid tumor, Ridgway osteogenic sarcoma, which is sensitivie to many established chemotherapeutic agents, did not respond to PALA. Daily or intermittent treatment with PALA did not significantly increase the life-span of mice bearing i.p. leukemia L1210. The survival time of mice bearing i.p. P388 leukemia was prolonged by PALA treatment by up to 64%. In a number of experiments mice bearing i.p. B16 melanoma survived 77 to 86% longer than did controls when treated with PALA (490 mg/kg) on Days 1, 5, and 9. Lewis lung carcinoma, a tumor refractory to most established antineoplastic agents, was highly sensitive to PALA. Treatment on Days 1, 5, and 9 following s.c. implantation of Lewis lung carcinoma was curative to 50% of the mice. If treatment was delayed until s.c. Lewis lung tumors had reached about 500 mg, PALA neither cured the mice nor produced significant tumor regression. However, extensive delay of tumor growth and prolongation of survival were still observed.

Mechanism of resistance of a variant of P388 leukemia to L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin).

Acivicin [L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; NSC 163501] is a fermentation-derived amino acid antibiotic antagonistic to L-glutamine which exhibits potent oncolytic properties. We have developed a variant of P388 leukemia resistant to acivicin (P388/ACIA) and compared its properties with those of the parent line (P388/S). An examination of the enzymes utilizing L-glutamine revealed that the basal specific activities of L-asparagine synthetase and L-glutaminase were 1-to 3-fold higher in the parent line. The activities of carbamoyl phosphate synthetase II, L-asparagine synthetase, formylglycinamide ribonucleotide amidotransferase, and guanosine monophosphate synthetase were about equally inhibited in the two cell lines, while there was a partial inhibition of 5-phosphoribosyl-1-pyrophosphate amidotransferase, fructose-6-phosphate amidotransferase, and L-glutaminase activities, found only in the sensitive line. Cytidine triphosphate synthetase activity was not inhibited in either line. There was no difference in the dose response or restitution of L-glutamine utilizing enzyme activities between the two lines. Acivicin treatment produced a 2- to 3-fold augmentation of the L-glutamine pools only in the sensitive line. Drug injection induced increased 5-phosphoribosyl-1-pyrophosphate levels in both lines. Acivicin perturbed guanosine nucleotide pools only in the sensitive line, indicating that the primary mechanism of action of acivicin in P388 leukemia may be directed at guanosine monophosphate synthetase. Transport studies demonstrated a restricted uptake of acivicin by the resistant cells. These studies suggest that the transport of acivicin and L-glutamine plays an important role in determining the sensitivity or resistance to acivicin in these tumors.

Synergistic cell killing by ionizing radiation and topoisomerase I inhibitor topotecan (SK&F 104864).

Topotecan (SK&F 104864), a water-soluble analogue of the topoisomerase I inhibitor camptothecin, is currently in Phase II clinical trial for solid tumors. We have characterized topotecan in terms of its effect upon gamma-radiation-induced cell killing. In colony formation experiments, subtoxic concentrations of topotecan (2 microM) potentiated radiation-induced killing of exponentially growing Chinese hamster ovary or P388 murine leukemia cultured cells. Survival curve shoulders were reduced; the slopes of the exponential portions of the curves were decreased to a small extent. D37 and D10 (radiation dose resulting in 37 and 10% survival of colony-forming ability) values were reduced by approximately 60 and 50%, respectively, in the case of Chinese hamster ovary cells. In P388 cells, topotecan reduced D37 by 35 to 40% and D10 by 20 to 25%. Potentiation of radiation-induced cell killing by topotecan was absolutely dependent upon the presence of the topoisomerase I inhibitor during the first few (less than 30) min after irradiation. Association of topoisomerase I with this effect was confirmed in studies of Chinese hamster ovary cells previously made resistant to camptothecin (and cross-resistant to topotecan), resulting in decreased cellular content of topoisomerase I. These cells were found to be 2- to 3-fold hypersensitive to gamma-radiation-induced killing. P388 camptothecin-resistant cells were further sensitized to the lethal effects of ionizing radiation by nontoxic treatment with the topoisomerase II inhibitor novobiocin, consistent with increased dependence of topoisomerase I-deficient cells upon topoisomerase II.

Rapamycin selectively inhibits the growth of childhood rhabdomyosarcoma cells through inhibition of signaling via the type I insulin-like growth factor receptor.

We show that cell lines derived from childhood alveolar rhabdomyosarcoma (RMS) are very sensitive to the growth-inhibitory effects of the immunosuppressive agent rapamycin (RAP), compared to other human cell lines (50% inhibitory concentration range of 0.1-8 ng/ml, compared to 1280 to > 10,000 ng/ml). Our data suggest that the sensitivity of RMS lines is due to RAP inhibition of insulin-like growth factor 1 receptor-mediated signaling, which is essential for continued proliferation of RMS cells. The embryonal RMS line Rh1, which was resistant to RAP in serum-containing medium (50% inhibitory concentration, 4180 ng/ml), was highly sensitive under autocrine conditions of growth, indicating that resistance was due to paracrine signaling pathways insensitive to RAP action. FK506 reversed RAP action in all cell lines, indicating a dependence on complexing with the cytosolic FK506-binding protein for activity.

Enhancement of antitumor activity of alkylating agents by the radiation sensitizer misonidazole.

The influence of the radiosensitizer misonidazole on the effectiveness of several alkylating agents and cis-platinum against advanced solid murine tumors was investigated. Tumor regrowth delay, frequency of tumor regressions, and animal life span were used to evaluate misonidazole in combination with cyclophosphamide, L-phenylalanine mustard, 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl)-1-nitrosourea, aziridinyl-benzoquinone, and cis-platinum. In the advanced M5076 ovarian carcinoma, misonidazole enhanced the activity of cyclophosphamide, L-phenylalanine mustard, 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl)-1-nitrosourea, and aziridinyl benzoquinone, but not cis-platinum. In early B16 melanoma, misonidazole plus cyclophosphamide was no more effective than cyclophosphamide alone. In advanced Lewis lung carcinoma, misonidazole enhanced the antitumor activity of cyclophosphamide but not 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl)-1-nitrosourea. Misonidazole, at 1000 mg/kg, increased the antitumor effectiveness of L-phenylalanine mustard and cyclophosphamide in M5076 tumors by factors of 2.2 and 1.8, but caused only a 1.2- and 1.3-fold increase in the myelotoxicity of these agents as determined by spleen colony assay of normal bone marrow. Misonidazole also increased the toxicity of cyclophosphamide and L-phenylalanine mustard in non-tumor-bearing mice but to a lesser degree than it enhanced antitumor activity. These results indicate that misonidazole is capable of enhancing the effects not only of ionizing radiation but of alkylating agents as well.