Steroid hormone receptor studies in melanoma model systems

The Transplantable B-16 melanotic melanoma carried in syngeneic C57B1/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrosedensity gradient centrifugation of cytosol after incubation with [³H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5–35 fmoles per mg cytosol protein. Scatchard analysis of [³H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 × 10^?10 M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [³H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [³H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high affinity sites with a dissociation constant of 2 × 10^?9 M. Binding levels from 70–610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.     

An improved culture medium for mouse blastocysts

Summary Eagle's basal medium, modified to contain essential amino acids at the concentrations optimal for mouse blastocyst hatching, attachment, and outgrowth, supported in vitro development of the mouse blastocyst better than other tissue culture media tested. This medium was improved for growth and differentiation of the inner cell mass by doubling the concentration of amino acids and glucose and by adding uridine (10⁻⁵ M) and β-mercaptoethanol (10⁻⁵ M). In this improved medium nearly all blastocysts grown from the two-cell stage hatched and formed trophoblast outgrowths, and 62% developed into two-layer egg cylinders. This work was supported by the U.S. Department of Energy.     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.     

Thymidine kinase activity in mouse embryo fibroblast cells infected with murine cytomegalovirus

Thymidine kinase activity was found in whole cell extracts of growing and stationary mouse embryo fibroblast cells after infection with murine cytomegalovirus. Determination of the kinetic constants and heat stability characteristics indicated that the enzyme activity from infected cells was different to that found in uninfected cells in the growth phase. The expression of thymidine kinase activity during virus replication was reflected by the incorporation of (6-³H) thymidine into acid precipitable fractions of infected cell cultures. Preliminary data from kinetic studies showed a reduction in the phosphorylation of thymidine by this enzyme activity in the presence of Acyclovir, a potent inhibitor of herpes virus replication.     

A mutation affecting the lactate dehydrogenase locus Ldh-1 in the mouse—I. Genetical and electrophoretical characterization

(101/El × C3H/El)F₁ male mice were injected intraperitoneally with the mutagen procarbazine hydrochloride and immediately caged with untreated test-stock females. Crude liver extracts from the offspring were subjected to polyacrylamide gel isoelectric focusing, and the gels were stained for six enzymes. In the experimental group (mutagen treated spermatogonial germ-cell stage), a dominant inherited banding alteration of the lactate dehydrogenase (LDH) pattern was detected. By crossing the heterozygous mutants, homozygotes were obtained that showed much less gel staining intensity. The mutation is codominantly expressed with 100% penetrance. The banding alteration was also observed in muscle, kidney, heart, blood, brain, testis, spleen, and lung. Polyacrylamide gel electrophoresis was performed with all the tissues examined. The mutation causes the intensity of the band corresponding to LDH-A (primary molecular form in muscle) to decrease from that of the wild type, while the intensity of the bands corresponding to LDH-B (primary molecular form in heart) remains constant. It is concluded that the mutation affects the locus coding for LDH of the muscle type. Ldh-1 ^c is proposed as the allele symbol.     

Linkage of Pgm-3 in the house mouse and homologies of three phosphoglucomutase loci in mouse and man

The discovery of a third phosphoglucomutase locus (Pgm-3) in the house mouse is reported. Three alleles are recognized on the basis of differences in electrophoretic mobility and enzymatic activity. Pgm-3 ^a (fast mobility and high activity) is present in inbred strain C57BL/10J and 24 other strains; Pgm-3 ^b (slow mobility and high activity) is present in LP/Pas and six other strains; and Pgm-3 ^c (no detectable activity in any tissue tested) is present in strain DBA/2J and 14 other strains. Seventy-four recombinant inbred strains derived from progenitors that differed at Pgm-3 were used to study genic linkage. Pgm-3 is on chromosome 9 and is linked to Sep-1, d, Mod-1, and Ltw-3. Gene order and recombination frequencies are estimated as d 3.8±1.8% Pgm-3 2.3±1.2% Mod-1. Substrate specificities and cofactor requirements show that mouse Pgm-1 is homologous with human Pgm-2, mouse Pgm-2 with human Pgm-1, and mouse Pgm-3 with human Pgm-3.This research was supported in part by NIH Research Grant GM18684 from the National Institute of General Medical Sciences to B.A.T. and by grants from NIH A105531-02 and the Volkswagon Foundation to Jan Klein. J.H.N. was a recipient of a Fellowship from the Max-Planck-Gesellschaft, Munich. G.S. and J.K. were supported by funds from the Deutsche Forschungsgemeinschaft.     

An allele (Pk-1 b ) from wild-caught mice that affects the activity and kinetics of erythrocyte and liver pyruvate kinase

A true breeding strain was made from a wild-caught mouse with low erythrocyte pyruvate kinase (E.C. 2.7.1.40) activity. This variation showed additive inheritance and segregated as an allele at a single locus (Pk-1 ^b). Mice homozygous for the reduced blood pyruvate kinase activity cosegregated for reduced liver activity. In both these tissues the variant enzyme had a lowered heat stability and reduced K _m values for ADP. An increased stimulation by FDP was also detected in the liver pyruvate kinase. No difference in the isoelectric point of the variant enzyme in either erythrocyte or liver was observed when compared with the enzyme from C57BL mice (Pk-1 ^a/Pk-1 ^a). It is concluded that Pk-1 is the structural gene for the erythrocyte and the major liver pyruvate kinase. No other tissue pyruvate kinase showed altered characteristics.This work was supported by a Medical Research Council grant.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

Genetic variability for regional brain gangliosides in five strains of young mice

The quantitative and qualitative distributions of gangliosides were determined in the cerebrum, cerebellum, and brain stem of five inbred strains (C57BL/6J, DBA/2J, LG/J, C3H/HeJ, BALB/cJ) of mice at 21 days of age. Genetic differences were found among the strains for wet weight, absolute amount of gangliosides per region, and concentration of ganglioside (expressed on both a wet and a dry weight basis) in all three regions of the brain. The water content of the various brain regions showed the least amount of genetic variability. Coefficients of genetic determination were used to estimate the magnitude of genetic influence on these traits in each brain region. Significant differences were also found among the five strains for the distribution of certain gangliosides. The DBA strain, which is susceptible to audiogenic seizure at this age, had the highest level of the myelin-enriched ganglioside G_M1 in all brain regions. Most of the genetic variation that influences the content and distribution of gangliosides among neurologically normal mice can be considered polygenic. Several possible sources of this genetic variation that may contribute to the differences observed among the strains are discussed.This work was supported by USPHS Grant NS 11853 and by a grant from the Swebilius Fund. T. N. S. is the recipient of a USPHS postdoctoral fellowship (1F32NS0443).