Melatonin inhibition of cancer growth in vivo involves suppression of tumor fatty acid metabolism via melatonin receptor-mediated signal transduction events.

The growth of rat hepatoma 7288CTC in vivo is stimulated by the uptake of linoleic acid (LA) and its metabolism to 13-hydroxyoctadecadienoic acid (13-HODE), an important mitogenic signaling molecule within this tumor. Conversely, the growth of a variety of experimental cancers in vivo is inhibited by either physiological or pharmacological levels of the pineal gland hormone melatonin, although the mechanism(s) are unknown. We tested the hypothesis that the mechanism of melatonin's anticancer action in vivo involves the inhibition of tumor LA uptake and metabolism to 13-HODE in hepatoma 7288CTC. Tumor uptake of LA and release of 13-HODE, measured in tissue-isolated rat hepatoma 7288CTC at 4-h intervals over a 24-h period, were highest during the light phase and lowest during the mid-dark phase, when plasma melatonin levels were lowest and highest, respectively. Pinealectomy eliminated this rhythm of tumor LA uptake and 13-HODE production, indicating that it was driven by the circadian melatonin rhythm. Perfusion of tissue-isolated tumors in situ with melatonin (1 nM) rapidly and reversibly inhibited the uptake of plasma fatty acids (FAs), including LA, and its metabolism to 13-HODE. These inhibitory effects of melatonin on tumor FA uptake and 13-HODE release were completely reversed by perfusion of tumors in situ with melatonin receptor antagonist S-20928, pertussis toxin, forskolin, or 8-bromo-cAMP. Perfusion of tumors in situ with melatonin also decreased tumor [3H]thymidine incorporation and DNA content; these effects on DNA synthesis were also prevented by the coperfusion of tumors with melatonin and S-20928, pertussis toxin, forskolin, 8-Br-cAMP, or 13-HODE. Pinealectomy stimulated tumor growth, LA uptake and metabolism to 13-HODE, and FA storage in hepatoma 7288CTC, whereas melatonin administration (200 microg/day) was inhibitory in vivo. Northern blot analysis revealed that, compared with normal liver tissue, hepatoma 7288CTC overexpressed mRNA transcripts for a plasma membrane-associated FA transport protein (FATP). FATP mRNA expression was unaffected by the treatment of tumor-bearing rats with daily afternoon melatonin injections or exposure to constant light. These results support a novel mechanism of tumor growth inhibition by melatonin involving a melatonin receptor-mediated suppression of cAMP levels, resulting in diminished tumor FA transport, possibly via decreased FATP function. The inhibition of these signal transduction events by melatonin culminates in the suppression of LA uptake, LA metabolism to the mitogenic signaling molecule 13-HODE, and cancer growth.

Unaltered class II histocompatibility antigens and pathogenesis of IDDM in BB rats.

The BB rat spontaneously develops insulin-dependent diabetes mellitus (IDDM) as an autoimmune abnormality involving the class II molecules of the major histocompatibility complex (MHC). The rat MHC (RT1 complex) encodes two class II loci, RT1.B and RT1.D. The possibility that variant or unique class II MHC molecules may be associated with IDDM susceptibility was directly examined by determining the nucleotide sequences of class II mRNAs and/or cDNAs from the diabetes-prone (DP) BB rat, the diabetes-resistant (DR) BB rat, the normal histocompatible Wistar-Furth (WF) rat, and the Lewis rat. Sequence analysis indicates that the beta-chains of the RT1.B and RT1.D molecules of the u haplotype from DP-BB, DR-BB, and WF rats are identical but that they are different from other rat alleles and published mouse class II sequences. At the nucleotide level, the NH2-terminal domain of RT1.D beta of the BB and WF rats differs by a single silent nucleotide substitution. Comparisons with the sequences of the Lewis rat indicate hypervariable allelic differences and that the u and I haplotypes are remarkably similar. These findings establish that the class II molecules of the DP-BB rat are not variant or unique and that unaltered class II molecules of the u haplotype support the autoimmune response in the BB rat.

Elevated mRNA levels of major histocompatibility complex class II genes in lymphocytes of autoimmune BB rats.

The BB rat spontaneously develops autoimmune abnormalities such as insulin-dependent diabetes mellitus and thyroiditis. The autoimmunity of the BB rat is controlled in part by genes of the major histocompatibility complex (MHC), known as the RT1 complex in the rat, and accumulating evidence suggests the involvement of MHC class II molecules. The RT1 complex specifies two types of class II molecules, which are encoded by the loci RT1.B and RT1.D. We have determined the relative steady-state mRNA levels of the class II genes RT1.B beta, RT1.D alpha, and RT1.D beta in splenic lymphocytes from individual autoimmune BB rats of various ages and from age-matched histocompatible normal Wistar-Furth (WF) rats. The relative steady-state mRNA levels of the RT1.D alpha and RT1.D beta genes, but not of the RT1.B beta gene, were elevated approximately 2.5-fold in lymphocytes of prediabetic BB rats 45-75 days old in comparison with age-matched normal WF rats and older BB rats greater than 75 days old. In the diabetic and nondiabetic BB rats greater than 75 days old, the RT1.D alpha and RT1.D beta transcripts were found at lower normal levels, similar to that of WF rats. In contrast, the RT1.B beta transcripts were found at comparable levels in lymphocytes of the BB and WF rats at all ages examined. The increased steady-state mRNA levels of the RT1.D alpha and RT1.D beta genes in the prediabetic BB rats may reflect differences in the proportion of lymphocytes expressing these genes and thus differences in splenic lymphocyte populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Restoration of abated T cell stimulation activity of mature dendritic cells.

Freshly isolated and cultured mature dendritic cells (DC) rapidly lose their high capacity to activate T cells. This diminished T cell activation activity is due, in part, to dramatically reduced expression of the costimulation ligands B7-1 (CD80) and B7-2 (CD86). Here we show that cultivation of mature DC with the lymphokines TNFalpha, GM-CSF, or TNFalpha plus GM-CSF, fully restored and enhanced their T-cell stimulation activity by two to threefold, four to fivefold, or six to sevenfold, respectively. The restored T-cell stimulation activity was directly correlated with induced and increased levels of CD80 (16-fold) and CD86 (10-fold) expression by the lymphokine-cultured DC. These results should facilitate many current DC-vaccination efforts towards generating more potent T cell responses.

Biologically active recombinant rat granulocyte macrophage colony-stimulating factor produced in Escherichia coli.

Rat granulocyte macrophage colony-stimulating factor (rGM-CSF) cDNA was amplified and cloned, and recombinant-rGM-CSF (R-rGM-CSF) was expressed and isolated from Escherichia coli. The synthesis of R-rGM-CSF was directed by a modified, inducible maltose binding protein (MBP) gene fusion expression vector, pMTR-23, and secreted to the periplasm. The vector pMTR-23 contains a new multiple cloning site encoding a unique thrombin-sensitive cleavage site and short spacer arm which facilitates separation of the MBP from the foreign protein domain of hybrid proteins. Biologically active R-rGM-CSF was rapidly purified by a combination of affinity and ion exchange chromatography, with a yield of 1.5 mg of R-rGM-CSF per liter of cultured cells. The purified R-rGM-CSF, like the native molecule, exhibits potent biological activity in two rGM-CSF-specific assays, considerably enhancing the accessory activity of rat dendritic cells and stimulating the differentiation of dendritic cells from fresh cultures of rat bone marrow cells. Although dendritic cells are difficult to isolate from tissues, the availability of R-rGM-CSF should now facilitate the development of large numbers of dendritic cells and the understanding of their regulatory role in the immune response.

Isolation of a recombinant lambda phage carrying nusA and surrounding region of the Escherichia coli K-12 chromosome.

A recombinant bacteriophage lambda, lambda argG-6, has been isolated which carries the argG gene and neighbouring loci on an EcoRI-generated 15.5 Kb DNA fragment from the Escherichia coli chromosome. The locations of the argG, nusA and pnp genes on the 15.5 Kb DNA fragment have been determined. In the case of nusA, a Tn5 insertion and sub-cloning of restriction fragments were used to locate the gene. The gene products of nusA and pnp have been identified on one- and two-dimensional polyacrylamide gels. The clockwise gene order was found to be argG-nusA-pnp.

Bacteriophage lambda vehicle for the direct cloning of Escherichia coli promoter DNA sequences: feedback regulation of the rplJL-rpoBC operon.

A derivative of bacteriophage lambda, lambda 21, has been constructed and used for the cloning of Escherichia coli DNA fragments carrying promoters. Phage lambda 21 lacks the lac promoter operator and can accept DNA fragments up to 9.8 kilobases in size at a unique HindIII restriction endonuclease site adjacent to lacZ. Recombinant phage that carry promoters are readily identified by their expression of lacZ. Lysogens of these phages in strains harboring a deletion of the chromosomal lac operon are capable of growth on lactose as sole carbon source and can be used to study some of the regulatory signals that act upon the cloned promoter. In principle, lambda 21 can be used to clone any promoter DNA sequence with HindIII termini. PJ, the primary promoter for the rplJL-rpoBC operon, and P beta, a weak promoter for rpoBC, have been cloned in lambda 21. Transcription of lacZ from PJ was found to be subjected to feedback control by ribosomal protein L10 and to a lesser extent by ribosomal protein L7/L12. This suggests a possible L10-binding site near PJ that regulates transcription from that promoter. Lysogens of the phage that carries P beta responded to two regulatory signals: a rho-sensitive termination site preceding rpoBC and induction of beta-galactosidase synthesis by rifampicin. This suggests that P beta is a bona fide promoter for rpoBC.

The complete sequence of the MHC class II chain RT.1D alpha u of the diabetic BB rat: mRNA levels of RT1.D alpha in lymphocytes.

The major histocompatibility complex of the rat (RT1 complex) encodes two sets of class II molecules referred to as RT1.B and RT1.D. The complete structure of the RT1.D alpha u chain of the diabetes prone BB rat was determined by the isolation and characterization of a full size cDNA. Comparisons of the nucleotide and protein sequences of RT1.D alpha with the analogous molecules, H-2 I-E alpha and HLA DR alpha, revealed that these alpha chains have been highly conserved during evolution. Southern blot analysis indicated an association of the RT1 haplotypes, 'u' and 'l', with Bam H1 DNA bands of 9.8 kb and 11.7 kb, respectively. The BB rat develops insulin dependent diabetes as an autoimmune abnormality. Accumulating evidence suggests a cellular mediated etiology and the involvement of class II molecules. The steady state levels of RT1.D alpha mRNA were measured in splenic lymphocytes of diabetes prone BB rats and age matched histocompatible normal nondiabetic WF rats by a RNase protection assay. Compared to WF rats, elevated transcripts of RT1.D alpha were found in lymphocytes of young BB rats (approximately 4x and approximately 2.5x greater at 20-40 and 40-75 d, respectively). In lymphocytes of older diabetic and nondiabetic BB rats (greater than 75 d) the levels of RT1.D alpha mRNA were lower than in the young BB rats and were found at the WF control levels. The increased steady state RT1.D alpha mRNA levels in the young BB rats may reflect differences in the proportion of splenic lymphocytes expressing this gene (activated lymphocytes), and thus differences in splenic lymphocyte populations. The steady state RT1.D alpha mRNA levels in lymphocytes of the normal rats were found to be relatively similar at all ages examined. The increased class II gene transcripts found in lymphocytes of young BB rats indicates that they possess a highly activated immune system.

Sequence and gene transfer analyses of HLA-CwBL18 (HLA-C blank) and HLA-Cw5 genes. Implications for the control of expression and immunogenicity of HLA-C antigens.

Our previous studies suggested that a serologically undetectable HLA-C blank allele (HLA-CwBL18) is either a variant Cw5 allele or a novel HLA-C Ag. To examine these possibilities, the CwBL18 and Cw5 genes from the TCC (HLA-A1, -A2, -B52, -B18, -Cw-, -Cw-) and QBL (HLA-A26, -B18, -Cw5) EBV-transformed B lymphoblastoid cell lines (LCL) were cloned, sequenced, and transferred into HLA-A, -B, -C null LCL mutant .221 cells. The CwBL18 Ag was detected on the cell surface of CwBL18 transferents by flow cytometry with the anti-class I mAb W6/32 but not by complement-mediated cytotoxicity with currently available HLA-C specific antisera. Sequence analysis of the Cw-BL18 gene indicated that the CwBL18 Ag is "C"-like because it contains all C-locus-specific residues and amino acid replacements commonly found in HLA-C alleles. However, the amino acid sequence of the CwBL18 Ag is unusual; CwBL18 lacks unique allele-specific residues when compared with the sequences of other HLA-C alleles. Moreover, apart from the C-locus-specific differences, the sequence of CwBL18 is identical to the HLA class I consensus sequence. This striking homology of CwBL18 to other HLA class I alleles suggests that CwBL18 may be a weak Ag. Taken together, these data demonstrate that CwBL18 is not a variant Cw5 Ag but is a newly described HLA-C Ag. In contrast to CwBL18, the Cw5 Ag is serologically detectable on the cell surface of Cw5 transferents with HLA-specific allo-antisera. Rather unexpectedly, Cw5 was usually expressed at a lower level than CwBL18 on the surface of .221 transferents as evaluated by W6/32 mAb binding analyses. The sequence of Cw5 revealed several unique amino acid replacements. Two of these substitutions, at residue 35 of the alpha 1 domain and residue 275 of the transmembrane domain, may be responsible for the reduced cell surface expression of Cw5. Additional unique replacements at residues 138 and 177 of the alpha 2 domain suggest that these amino acids may be important in the formation of an epitope recognized by a Cw5-specific antibody.

New actions of melatonin on tumor metabolism and growth.

Melatonin is an important inhibitor of cancer growth promotion while the essential polyunsaturated fatty acid, linoleic acid is an important promoter of cancer progression. Following its rapid uptake by tumor tissue, linoleic acid is oxidized via a lipoxygenase to the growth-signaling molecule, 13-hydroxyoctadecadienoic acid (13-HODE) which stimulates epidermal growth factor (EGF)-dependent mitogenesis. The uptake of plasma linoleic acid and its metabolism to 13-HODE by rat hepatoma 7288CTC, which expresses both fatty acid transport protein and melatonin receptors, is inhibited by melatonin in a circadian-dependent manner. This inhibitory effect of melatonin is reversible with either pertussis toxin, forskolin or cAMP. While melatonin inhibits tumor linoleic acid uptake, metabolism and growth, pinealectomy or constant light exposure stimulates these processes. Thus, melatonin and linoleic acid represent two important environmental signals that interact in a unique manner to regulate tumor progression and ultimately the host-cancer balance.

Activation of cation transport by lymphokines in B cells without induction of DNA synthesis or immunoglobulin gene transcription.

We report on an experimental model that permitted us to evaluate the biologic relevance of membrane-associated biochemical events with respect to cell proliferation and maturation, each induced by distinct sets of signals. Antigen-affinity-enriched murine B cells cultured in the presence of a proliferative signal induced by LPS showed activation of Na+/K+ ATPase and enhanced the uptake of proline, followed by RNA, protein, and DNA synthesis, without the generation of antibody. Stimulation with both the proliferative signal(s) and the maturation signal(s) derived from lymphokines of an EL-4 thymoma induced B cells to proliferate and synthesize mRNA encoding mu-chain of IgM and to mature into IgM-secreting cells. Most important, the secretory product of EL-4, in the absence of LPS, activated Na+/K+ ATPase but failed to stimulate uptake of proline and synthesis of DNA or mu-specific mRNA. A similar response was observed in splenocytes depleted of T cells and in unfractionated spleen cells. Thus a component secreted by EL-4 can induce some of the early molecular events characteristic of the proliferative response but lacks the ability to initiate blast transformation and DNA synthesis.

X-ray and primary structure of horse serum albumin (Equus caballus) at 0.27-nm resolution.

The amino-acid sequence and three-dimensional structure of equine serum albumin have been determined. The amino-acid sequence was deduced from cDNA isolated from equine liver. Comparisons of the primary structure of equine serum albumin with human serum albumin and bovine serum albumin reveal 76.1% and 73.9% sequence identity, respectively. The three-dimensional structure was determined crystallographically by the molecular-replacement method using molecular coordinates from the previously determined structure of human serum albumin, to a resolution of 0.27 nm. In accordance with the primary structure, the three-dimensional structures are highly conserved. There is a root-mean-square difference between alpha-carbons of the two structures of 0.201 nm. The association constants (Ka) for the binding of 2,3,5-triiodobenzoic acid were determined by ultrafiltration methods for equine and human serum albumins to be approximately 10(4) M-1 and 10(5) M-1, respectively. Crystallographic studies of equine serum albumin reveal two binding sites for 2,3,5-triiodobenzoic acid identical with those previously reported for human serum albumin which are located within subdomains in IIA and IIIA. Details and comparisons of the binding chemistry are discussed.