Recombinant human tumor necrosis factor induces acute reductions in food intake and body weight in mice.

We examined the effects of treatment with rHuTNF on food consumption and body weight in C3H/HeJ mice. rHuTNF was administered intraperitoneally either by injections of 3, 12, or 24 micrograms twice a day or by implantation of osmotic pumps that released 0.75, 3, or 12 micrograms per day. Dose-dependent reductions in both food intake and weight were induced by rHuTNF. However, in spite of continued exposure to rHuTNF, the mice developed a resistance to rHuTNF and resumed their pretreatment food intake and weight. Non-immunological factors may play a role in the development of this tolerance, since it developed rapidly and faded within 2 wk of cessation of exposure to rHuTNF.

5-Aminouracil treatment. A method for estimating G2.

Treatment of Vicia faba lateral roots with a range of concentrations of 5-aminouracil (5-AU) indicate that cells are stopped at a particular point in interphase. The timing of the fall in mitotic index suggests that cells are held at the S - G(2) transition. When cells are held at this point, treatments with 5-AU can be used to estimate the duration of G(2) + mitosis/2 of proliferating cells. Treatment with 5-AU can also be used to demonstrate the presence of subpopulations of dividing cells that differ in their G(2) duration. Using this method, 5-AU-induced inhibition, we have confirmed that in V. faba lateral roots there are two populations of dividing cells: (a) a fast-dividing population, which makes up approximately 85% of the proliferating cell population and has a G(2) + mitosis/2 duration of 3.3 hr, and (b) a slow-dividing population, which makes up approximately 15% of dividing cells and has a G(2) duration in excess of 12 hr. These estimates are similar to those obtained from percentage labeled mitosis (PLM) curves after incorporation of thymidine-(3)H.

Tumor necrosis factor not detectable in patients with clinical cancer cachexia.

Tumor necrosis factor (TNF) has been implicated in the pathogenesis of cachexia in neoplastic and infectious diseases. To assess the relationship between TNF and weight loss among cancer patients, we assayed TNF levels in serum from 19 patients who had lost 8%-40% of premorbid weight. The weight loss experienced by these patients was not attributable to anticancer therapy, gastrointestinal disorders, or other medical problems. TNF was measured using a quantitative sandwich enzyme-linked immunosorbent assay that is sensitive to human TNF in serum at concentrations greater than or equal to 40 pg/mL. No TNF was detected in serum samples from the 19 patients studied.

An analysis of the binding of the chick oviduct progesterone-receptor to chromatin.

The binding of progesterone-receptor complexes to chromatin from target and nontarget tissues was studied in vitro. Chromatin from both target and nontarget tissues responds in a similar manner to saly and cofactors and has the same K(D) (approx. 3.10(-9) M) for the progesterone-receptor complex. The only observed difference in the binding of the progesterone-receptor complex to target and nontarget chromatins is the difference in total number of acceptor sites. oviduct chromatin has approx. 1300 sites/pg DNA, spleen chromatin has approx. 840 sites/pg DNA, and erythrocyte chromatin has about 330 sites/pg DNA. The K(D) and number of acceptor sites for progesterone-receptor complex binding to oviduct chromatin remains the same even after extensive purification of the progesterone-receptor complex. Activation of cytosol labeled with [3H]progesterone by preincubation at 25 degrees C, analogous to that required for maximal nuclear binding, occurs if the binding studies to chromatin are performed in 0.025 M salt. The absence of an observable temperature effect when the studies are performed at 0.15 M salt is due to the activation of the receptor by salt. The dissociation of the progesterone-receptor complex from chromatin exhibits a single dissociation rate and the initial event is the appearance of free progesterone rather than a progesterone-receptor complex. Lastly, the treatment of chromatin with an antibody prepared against either single-stranded DNA or double-stranded DNA does not alter the extent of binding of the progesterone-receptor complex. Similarly, pretreatment of chromatin with a single-stranded nuclease does not inhibit the capacity of chromatin to bind the hormone-receptor complex.

Distribution binding sites for the progesterone receptor within chick oviduct chromatin.

Chromatin from the oviducts of estrogen-treated chicks was sheared and fractionated on sucrose gradients. This resulted in the production of several chromatin fractions which differ in their sedimentation properties, protein composition, the number of acceptor sites for the progesterone-receptor complex, and the ability to serve as a template for RNA synthesis. The pellet chromatin fraction shows an enhanced ability to bind the progesterone-receptor complex in vitro and in cell-free systems. Kinetic analysis indicates that the majority of the acceptor sites for the progesterone-receptor complex are located in the pellet chromatin fraction which is lowest in template activity. The sites may be important loci for initiating the changes in RNA synthesis following the exposure of target cells to steroid hormones.

Tumor necrosis factor and endotoxin can cause neutrophil activation through separate pathways.

We investigated the possibility that tumor necrosis factor (TNF) mediates neutrophil activation by endotoxin. The number of C3b receptors on the neutrophil cell-surface was used as the indicator of activation, as assessed by indirect immunofluorescence. Incubation of buffy-coat neutrophils with TNF-alpha for 30 minutes at 37 degrees C caused neutrophil activation, increasing C3b receptor-dependent fluorescence from 340 with buffer alone to 580 with TNF (250 pg/mL). Increasing amounts of anti-TNF IgG progressively inhibited neutrophil activation by TNF (250 pg/mL). Addition of the active dose range of anti-TNF to neutrophils incubating in endotoxin (10 ng/mL) did not affect the degree of endotoxin-mediated neutrophil activation. Mixtures of neutrophils with the 50% suppressive dose of anti-TNF and varying endotoxin concentrations showed the same degree of neutrophil activation as mixtures without the antibody. Thus, an antibody that can inhibit TNF-mediated neutrophil activation does not inhibit endotoxin-mediated neutrophil activation. We conclude that endotoxin and TNF can activate neutrophils through separate pathways.

Interleukin-1, endotoxin or tumor necrosis factor/cachectin enhance the level of plasminogen activator inhibitor messenger RNA in bovine aortic endothelial cells.

It is known that either endotoxin (LPS) or interleukin-1 (IL-1) increase the activity of plasminogen activator inhibitor (PAI) in the culture media of human and bovine endothelial cells. We have confirmed these results in bovine aortic endothelial cells (BAEC). To determine if this effect was mediated by increases in the level of PAI messenger RNA (mRNA) we examined the effects of these cytokines on PAI mRNA levels in BAEC, using RNA blot analyses. Treatment of BAEC with either IL-1, LPS, or human recombinant tumor necrosis factor/cachectin (TNF) dramatically increased the level of PAI mRNA. Since elevated levels of PAI will decrease fibrinolytic potential, this mechanism is in concert with the known increase in in vivo procoagulant potential induced by these agents and could contribute to thromboembolic phenomena.

Dysdifferentiative nature of aging: age-dependent expression of mouse mammary tumor virus and casein genes in brain and liver tissues of the C57BL/6J mouse strain.

Many aspects of the aging process could be the result of cells slowly drifting away from their proper state of differentiation. This possibility has been studied by searching for an age-dependent increase in the expression of specific genes in tissues where expression of these genes would not normally be expected. In these studies, cDNA probes of specific genes are used in a DNA X NA hybridization assay to detect possible complementary RNA sequences in tissues of different-aged animals. Using this technique in past experiments, a qualitative increase in the RNA sequence complexity of mouse leukemia virus (MuLV) and a quantitative increase in the amount of alpha- and beta-globin RNA were found with increasing age in the brain and liver of the C57BL/6J mouse strain. We report here a similar age-dependent qualitative increase in the RNA sequence complexity for mouse mammary tumor virus (MMTV) but no quantitative or qualitative age-dependent change in casein RNA sequences for the same tissues and mouse strain.

Anti-equine tumor necrosis factor (TNF) activity of antisera raised against human TNF-alpha and peptide segments of human TNF-alpha.

Antisera raised in rabbits against either purified recombinant-derived human tumor necrosis factor (TNF)- alpha (huTNF) or huTNF peptide-bovine thyroglobulin conjugates were evaluated for anti-equine TNF (eqTNF) activity. Binding and neutralizing anti-eqTNF activities were found in antisera raised against whole huTNF or against either of the peptides containing the N-terminal 15 amino acids of huTNF (huTNF[1-15] and huTNF[1-31]). Anti-eqTNF activity was not detected in antisera raised against huTNF[65-79], huTNF[98-111] or huTNF[124-141] peptides. The addition of excess huTNF[1-15] completely inhibited the ability of anti-huTNF[1-15] to bind eqTNF and reduced by approximately 25% the anti-eqTNF activity of an antiserum raised against whole huTNF. Nonconjugated huTNF[1-15] did not have eqTNF agonist or antagonist activity. Results were consistent with previous structural and functional data implicating the N-terminus of huTNF in receptor binding and indicate that the homologue of huTNF[1-15] on eqTNF may be a potentially important target for neutralizing anti-eqTNF antibodies.

Murine mammary tumor virus expression during mammary tumorigenesis in BALB/c mice.

Steady-state levels of murine mammary tumor virus (MuMTV) RNA were quantitated during mammary tumorigenesis in BALB/c mice by molecular hybridization with a representative MuMTV complementary DNA (cDNA) probe. Hyperplastic alveolar nodule (HAN) lines are preneoplastic mammary lesions that were induced in BALB/c mice by hormones alone or in combination with 7,12-dimethylbenz(a)anthracene and give rise to mammary tumors. The hormone-induced HAN lines D1 and D2 contained detectable amounts of hybridizable MuMTV sequences. MuMTV RNA sequences were also observed in five of the six transplanted BALB/c mammary tumors that were examined. Similar levels of hybridizable MuMTV RNA were observed between the D1 or D2 HAN line and mammary tumors derived from each HAN line. The D2 HAN line as well as D2, C4, and CD8 mammary tumors accumulated RNA that was apparently homologous to most of the MuMTV genome. Thermal denaturation of hybrids indicated extensive sequence homology between the MuMTV cDNA and hybridizable RNA in the BALB/c HAN lines and mammary tumors. A low level of type C viral RNA was observed in the BALB/c HAN lines and most mammary tumors by molecular hybridization with a cDNA to Moloney murine leukemia virus. These data demonstrate that MuMTV sequences are frequently expressed in hormone-induced BALB/c HAN lines and mammary tumors derived from HAN lines or ductal hyperplasias induced in BALB/c mice by hormones and/or a chemical carcinogen. The transition from the preneoplastic to the neoplastic state in BALB/c mice does not appear to be due to a change in the steady-state levels of MuMTV RNA since the hormone-induced HAN lines and mammary tumors had similar levels of hybridizable MuMTV RNA.

Hormonal regulation of murine mammary tumor virus RNA expression during mammary tumorigenesis in BALB/c mice.

The effects of glucocorticoids and prolactin on murine mammary tumor virus (MuMTV) RNA expression in preneoplastic outgrowth lines and mammary tumors in BALB/c mice were investigated. Hyperplastic alveolar nodules (HAN) and a ductal hyperplasia (DH) are induced in virgin BALB/c mice by prolonged hormonal stimulation or treatment with 7,12-dimethylbenz(a)anthracene or both. Mice bearing HAN or DH outgrowth lines and mammary tumors that arose from the outgrowth lines were treated with glucocorticoids or prolactin. MuMTV RNA was quantitated by hybridization with a representative complementary DNA probe specific for MuMTV RNA. Prolactin treatment did not increase MuMTV RNA in the BALB/c HAN or DH outgrowth lines or tumors. MuMTV RNA increased after glucocorticoid treatment in the C3, C4, and C5 HAN outgrowth lines and in tumors that arose from the D1, D2, C4, and C5 HAN and CD8 DH outgrowth lines. No increase in MuMTV RNA with glucocorticoid treatment was observed in the D1 or D2 HAN outgrowth line, in the CD8 DH outgrowth lines, and in tumors that arose from the C3 HAN outgrowth line. The ability of glucocorticoids to stimulate MuMTV expression was specific since the response was dose dependent and specific for glucocorticoid hormones. Glucocorticoid treatment did not increase the level of type C viral RNA in the majority of hormone- or 7,12-dimethylbenz(a)anthracene-induced HAN outgrowth lines or tumors. These observations suggested that glucocorticoids may influence MuMTV expression during mammary tumorigenesis in BALB/c mice.

Partial expression of endogenous mouse mammary tumor virus in mammary tumors induced in BALB/c mice by chemical, hormonal, and physical agents.

The possible interaction of environmental factors with the endogenous mouse mammary tumor virus (MMTV) genome in the development of mammary tumors in the low-tumor-incidence BALB/c mouse strain was examined. Tumors were induced in virgin female animals by treatment with chemical carcinogen 7,12- dimethylbenz[alpha]anthracene or urethan, with or without prolonged hormonal stimulation, or by X-irradiation. Concomitant hormonal stimulation resulted in increased tumor incidences compared with those induced by chemical carcinogen treatment alone. The frequency of tumor induction by irradiation alone or in combination with urethan or prolactin stimulation was very low. MMTV expression in the mammary tumors was assayed by nucleic acid hybridization and by immunohistochemical staining. Depending upon the treatment group, 0 to 89% of the tumors contained detectable levels of MMTV RNA (>/=0.0005% of the total cellular RNA). Tumors which contained detectable viral transcripts exhibited only low levels of MMTV RNA, which did not appear to represent the accumulation of RNA sequences homologous to the entire MMTV genome; synthesis of MMTV structural proteins was detected in only one tumor. Viral RNA-positive tumors were generally associated with a longer latent period. MMTV RNA expression occurred in tumors classified histologically as adenoacanthomas, as well as in mammary adenocarcinomas, although the cell types in the adenoacanthomas expressing viral RNA were not identified. It does not appear that expression of the endogenous MMTV genome is required for maintenance of all mammary tumors in BALB/c mice, although partial genome expression undetectable by the methods employed cannot be ruled out. Linear regression analyses were performed. The mean time to tumor appearance and the percentage of tumors which were MMTV RNA positive were found to vary linearly as a function of the total dose of 7,12-dimethylbenz[alpha]anthracene administered. The percentage of tumors which were MMTV RNA positive was also shown to be linearly related to the mean time to tumor appearance. These relationships provide a basis for predictions in the BALB/c system related to these parameters.

Detection of mouse mammary tumor virus RNA in BALB/c tumor cell lines of nonviral etiologies.

A complementary DNA (cDNA) probe to mouse mammary tumor virus (MMTV) RNA was synthesized using calf thymus DNA oligonucleotides as a random primer. This probe was then used to study the expression of MMTV RNA in cell lines from BALB/c tumors induced in vivo either spontaneously or in response to viral, chemical, or hormonal stimuli. The cDNA had a length of approximately 400 to 500 nucleotides and specifically hybridized to MMTV RNA and BALB/c lactating mammary gland RNA, but not to Moloney leukemia virus RNA. Calf thymus DNA-primed cDNA could protect 50% of iodinated MMTV RNA from S1 nuclease digestion at cDNA-RNA ratios of 1:1 and 90% of labeled viral RNA at ratios of 10:1. Thermal denaturation of MMTV RNA-cDNA hybrids yielded a T(m) of 88.5 degrees C, indicative of a well-base-paired duplex. Screening of mouse mammary tumor cells for MMTV sequences revealed that three out of five lines of BALB/c origin had undetectable levels of viral RNA (

Mouse mammary tumor virus genome expression in chemical carcinogen-induced mammary tumors in low- and high-tumor-incidence mouse strains.

Involvement of mouse mammary tumor virus (MMTV) in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis was investigated in low- (BALB/c) and high- (BALB/cfC3H) mammary-tumor-incidence mouse strains. Both strains contain endogenous MMTV integrated into the cellular genome. Additionally, BALB/cfC3H mice are infected with exogenous MMTV-S which is responsible for a higher incidence of mammary tumors in breeding females. Administration of DMBA to virgin mice of both strains resulted in a moderate frequency of mammary tumors within 40 wk after treatment. No differences were found in DMBA-induced tumor incidences at 18 wk (6% and 7%) or at 38 wk (29% and 36%) after treatment of BALB/c and BALB/cfC3H mice, respectively. Expression of MMTV in these tumors was examined by assaying for the presence of MMTV RNA by hybridization using MMTV-specific cDNA and by immunohistochemical staining utilizing antibodies against MMTV 52,000-dalton glycoprotein, gp52, and 28,000-dalton internal protein, p28. Of 16 BALB/c tumors assayed, 11 did not contain detectable levels of MMTV RNA and the remaining 5 tumors contained only low levels (0.0005-0.0010%) of viral RNA. Importantly, MMTV RNA was not detected in 5 of 27 BALB/cfC3H tumors. The other BALB/cfC3H tumors contained quantities of MMTV RNA ranging from 0.0006 to 0.4170%. Most BALB/cfC3H tumors with detectable levels of MMTV RNA also synthesized viral proteins gp52 and p28. Thus, expression of the complete MMTV genome is not requisite for maintenance of the tumor phenotype in DMBA-induced mammary tumors in either BALB/c or BALB/cfC3H virgin mice under 1 year of age.

Fractionation of chick oviduct chromatin. Nuclease-resistant deoxyribonucleic acid.

Chromatin isolated from several chick tissues was treated with micrococcal nuclease. A limited degree of tissue specificity of chromatin DNA resistance to nuclease digestion was observed. No difference in the extent of nuclease resistance of chromatin DNA was detected during oestrogen-induced oviduct differentiation. This suggested that the amount of non-histone chromosomal protein does not play an important role in the sensitivity of chromatin DNA to nuclease digestion. Studies of nuclease resistance of chromatin DNA after dissociation and reconstitution of chromatin proteins and ethanol extraction of chromatin indicate that the histones protect the DNA from nuclease attack. Slow thermal denaturation of nuclease-resistant DNA suggests that the protected DNA sequences may be (A+T)-rich, and the (G+C)-rich satellites present in total chick DNA are sensitive to nuclease.