Studies on the mechanism of 3-deazaguanine cytotoxicity in L1210-sensitive and -resistant cell lines

Summary 3-Deazaguanine (3-DG), a purine analogue, has unusual antitumor activity against experimental mammary tumor models and a number of other solid tumors. Others have shown that mutant CHO cells deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRTase) or adenine phosphoribosyl transferase (APRTase) are resistant to 3-DG. We developed a L1210 cell line resistant to 3-DG, L1210/3-DG, by subculturing the parent L1210/0 cells in the presence of increasing concentrations of 3-DG. The IC₅₀ was 3.5 M and 620 M for L1210/0 and L1210/3-DG, respectively. Cytotoxicity studies proved the resistance to be stable. Examination of the baseline-specific activity of HGPRTase and APRTase showed that the former was 118-fold lower in L1210/3-DG than in L1210/0, and the latter demonstrated no difference. A 4-h treatment of the cell lines at IC₅₀ doses showed 48% and 23% reductions in IMP dehydrogenase in L1210/0 and L1210/3-DG, respectively. The rate of de novo purine biosynthesis was studied by using [¹⁴C]formic acid. Formate flux increased 2-fold in L1210/3DG in concert with the observed deficiency of HGPRTase in the cell line. 3-DG uptake was studied with [¹⁴C]-labelled compound. The total radioactivity was 9-fold higher in L1210/0 than in L1210/3-DG at 2 h. Subsequent chromatographic separation of radioactivity showed the 3-DG and 3-deazaguanosine pools of the drug to be equal in both lines. However, 3-DG nucleotide pools at 1 min and 2 h were 2.5-fold and 16-fold lower, respectively, in L1210/3-DG than in L1210/0. 3-DG incorporation studies with radiolabelled drug demonstrated that 3-deazaguanine is incorporated in the acid-insoluble fraction of the cell. These studies conclude that HGPRTase, and not APRTase, is required for the activation of drug. Inhibition of IMP dehydrogenase is partially responsible for antitumor activity of the drug. The incorporation of drug into nucleic acids may be a major mechanism for its antitumor activity. Further studies using a cloned cDNA probe for hypoxanthine guanine phosphoribosyltransferase (HGPRT) demonstrated no change in the DNA arrangements of the L1210/3-DG cell line, and Northern blot analysis showed approximately equal expression of mRNA in both cell lines.Abbreviations used APRTase adenine phosphoribosyltransferase - HGPRTase Hypoxanthine guanine phosphoribosyltransferase - IMPD Inosine mono-phosphate dehydrogenase - PRPP 5-Phosphorylribose-1-pyrophosphate - AOPCP , -Methyleneadenosine 5-diphosphate - NAD Nicotineamide dinucleotide - EDTA Ethylenediamine tetra acetic acid Presented at annual meeting of American Association of Cancer Research in May, 1986Supported in part by Warner-Lambert Company, Ann Arbor, Michigan     

"Empty wall" and "vertical strut" signs of ACL insufficiency

We report our observation on the "empty wall" and "vertical strut" signs of anterior cruciate ligament (ACL) insufficiency. ACL tears most commonly occur in the midsubstance; arthroscopic evaluation of patients with these tears often reveals minimal evidence of previous ACL tissue along the intercondylar wall, thus giving the appearance of an "empty wall." In proximal ACL tears, the long remnant of ACL tissue may adhere to adjacent PCL tissue. Arthroscopically, one may see this vertically oriented strut of tissue, which to the casual arthroscopist may mimic a normal-appearing ACL except for orientation and tension. In addition, the "empty wall" sign will be noted because the lateral intercondylar wall becomes easily visible following ACL injury. In two separate prospective studies of 84 such patients, the combined incidence of the empty wall sign was 82%, and the incidence of the vertical strut sign was 50%. These findings should be sought for meticulously at the time of arthroscopic evaluation. The vertical strut should not be misinterpreted as an aberrantly oriented ACL or partial ACL tear.     

Expression of CD83 in the murine immune system

CD83 is used as a marker for mature dendritic cells (DC) in man. We have developed a new monoclonal antibody (mAb), Michel-17, that specifically recognizes mouse CD83. We show that murine CD83 is expressed mainly on mature DC and on activated T cells. Histological analysis of serial spleen sections revealed a CD83 expression pattern resembling that of MIDC-8, a known murine DC marker molecule. In contrast to other costimulatory receptors, cross-linking of CD83 with the mAb Michel-17 on DC or T cells does not induce any activation signals. Our data describe for the first time the expression pattern of murine CD83, which is comparable to that of human CD83.The unique mAb Michel-17 will help to elucidate the biological functions of the CD83 molecule in more detail.     

Structural model for T-cell recognition of HLA class II-associated alloepitopes.

In an effort to investigate the structure-function relationship of HLA class II molecules vis-à-vis alloepitope expression, cloned T-cell reagents were used to define polymorphic epitopes associated with DR and DQ molecules. DNA sequences of genes encoding allelic or isotypic DR or DQ molecules that appear to express the same T-cell-defined epitopes were compared in an attempt to identify association of shared sequences with shared epitopes. When sequence sharing is associated with shared epitope expression, we suggest that it is the shared sequence that encodes the epitope in question. Based on the hypothetical three-dimensional structure of the class II molecule, an approximation is made as to which parts of the HLA class II molecule are involved in alloepitope expression. T-cell clones were generated from cells primed against HLA-DR2 haplotypes representing the cellularly defined subgroups Dw2 or Dw21 (previously designated MN2, FJ0, or Tb24). Those clones determined to be DR- or DQ-directed based on monoclonal antibody inhibition assays were tested by panel cell analysis utilizing DR2-positive and DR2-negative target cells. The data support the concept that amino acids 67, 70, 71, and 74 for DR molecules and amino acids 57, 70, and 71 for DQ molecules, which appear to comprise one face of the alpha helix, are of primary importance in T-cell recognition. In other cases, sharing of both the second hypervariable region (amino acids 25-33) and the third hypervariable region (amino acids 67-74) appears necessary to explain epitope sharing for DR molecules. We emphasize that the involvement of these two hypervariable regions may indicate that alloepitope expression involves the complex of class II molecule plus peptide, with the second HVR primarily involved in determining which peptides are bound and the third in T-cell receptor (TcR) recognition and/or peptide binding; we do not rule out that conformational changes of the second HVR can induce conformational changes in the third HVR. Finally, shared alloepitopes detected by some clones could not be explained based on shared primary sequences.     

Single-cycle immunodeficiency viruses provide strategies for uncoupling in vivo expression levels from viral replicative capacity and for mimicking live-attenuated SIV vaccines

To reduce the risks associated with live-attenuated immunodeficiency virus vaccines, single-cycle immunodeficiency viruses (SCIVs) were developed by primer complementation and production of the vaccine in the absence of vif in a vif-independent cell line. After a single intravenous injection of SCIVs into rhesus monkeys, peak viral RNA levels of 10(3) to 10(4) copies/ml plasma were observed, indicating efficient expression of SCIV in the vaccinee. After booster immunizations with SCIVs, SIV-specific humoral and cellular immune responses were observed. Although the vaccine doses used in this pilot study could not protect vaccinees from subsequent intravenous challenge with pathogenic SIVmac239, our results demonstrate that the novel SCIV approach allows us to uncouple in vivo expression levels from the viral replicative capacity facilitating the analysis of the relationship between viral expression levels or viral genes and immune responses induced by SIV.     

Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1

The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.     

Strain differences in mouse cellular responses to Mycobacterium lepraemurium and BCG subcutaneous infections. I. Analysis of cell surface phenotype in local granulomas.

C57BL/6, BALB/c and CBA mice were subcutaneously infected with either Mycobacterium lepraemurium (MLM) or BCG, and studied for bacillary growth, granuloma size of infected footpads and draining lymph nodes (DLN), and DLN cell surface phenotype. Whereas, BCG-infected mice controlled the infection and developed early and large granulomas, MLM-infected mice exhibited major strain variations in their resistance to the infection, as well as in the granuloma size and kinetics. C57BL/6 mice, highly resistant, displayed early and regressive granulomas; BALB/c mice showed lower resistance and early granulomas that grew continuously; CBA mice, highly susceptible, developed late, soft, phagocyte-rich granulomas. Important strain differences in lymph node lymphocyte subset distribution could be observed prior to any infection: C57BL/6 mice displayed higher B cell percentages than both of the other strains and BALB/c mice showed the highest CD4/CD8 ratios, followed by CBA and C57BL/6 mice. BCG and MLM infections both induced similar changes of these parameters in all three strains: that is a decrease of the B cell percentage and a decrease of the CD4/CD8 ratio, and the strain differences observed in uninfected mice persisted. On the other hand, DLN cells stimulated by the infecting bacillus and interleukin 2 also displayed an increase of the CD8 T cell percentage as compared with normal lymph node cells, but this phenomenon was much less pronounced in BALB/c mice, whether infected by MLM or BCG, and in MLM-infected CBA mice, than in BCG- or MLM-infected C57BL/6 (B6) mice. Thus the ability of C57BL/6 mice to generate an early and persistent CD8 T cell response to mycobacteria may contribute to their resistance to MLM.     

Study of blood group B antigen with a specific monoclonal antibody (anti-B, b-183).

A murine anti-B monoclonal antibody was obtained by the hybridoma technique. This antibody called anti-B (b-183) is of IgM nature; it is capable of agglutinating normal B, B3, Bx, cis AB and some acquired B red cells. Its association constant is 1.1 X 10(8) l/mol, and appears high compared to those of the monoclonal anti-A. This monoclonal anti-B was used to determine the number of B sites on B3 and Bx red cells.     

Diagnosis of arylsulfatase A deficiency in intact cultured cells using a fluorescent derivative of cerebroside sulfate

A fluorescent derivative of cerebroside sulfate (12-(1-pyrene)dodecanoyl-sphingosylgalactosyl-0-3-sulfate (P12-sulfatide) has been synthesized as a potential substrate for the determination of cerebroside sulfatidase (or arylsulfatase A) activity. It was administered into cultured human skin fibroblasts and thereby utilized for the diagnosis of arylsulfatase A deficiency. Cultured skin fibroblasts from normal individuals and healthy persons suffering from a pseudoarylsulfatase A deficiency (PD) degraded the P12-sulfatide, while in cells derived from a metachromatic leukodystrophy (MLD) patient it remained essentially intact. This contrasts with in vitro determinations of enzymatic activity, where the MLD or PD-derived arylsulfatase A exhibit similar deficiency, in spite of a profoundly different clinical course. Administration of the fluorescent sulfatide into the intact cells permitted a sensitive and rapid diagnosis of MLD and its distinction from the PD-phenomenon. This might be of particular importance for cases in which a rapid diagnosis is required and for prenatal diagnosis of fetuses from families afflicted with both MLD and pseudo-deficiency mutant genes.