Eric H. Westin

Cloning and characterization of complementary DNA for human tryptase.

The amino acid sequence of human mast cell tryptase was determined from corresponding cDNA cloned from a lambda ZAP library made with mRNA derived from a human mast cell preparation. Tryptase is the major neutral protease present in human mast cells and serves as a specific marker of mast cells by immunohistologic techniques and as a specific indicator of mast cell activation when detected in biologic fluids. Based on nucleic acid sequence, human tryptase consists of a 244-amino acid catalytic portion of 27,423 D with two putative N-linked carbohydrate binding sites and a 30-amino acid leader sequence of 3,048 D. A His74, Asp120, Ser223 catalytic triad and four cystine groups were identified by analogy to other serine proteases. Regions of amino acid sequence that are highly conserved in serine proteases, in general, were conserved in tryptase. The catalytic portion of human tryptase had an 84% amino acid sequence similarity with that of dog tryptase; their leader sequences had a 67% similarity. Asp217 in the substrate binding pocket of human tryptase is consistent with a specificity for Arg and Lys residues at the site of cleavage (P1), whereas Glu245 is consistent with the known preference of human tryptase for substrates with Arg or Lys also at P3, analogous residues also being present in dog tryptase. Asp244, which is substituted for the Gly found in dog tryptase and in most serine proteases, is present in the putative substrate binding pocket and may confer additional substrate specificity on human tryptase for basic residues. Further studies now can be designed to elucidate these structure-function relationships.

In vitro effects of bryostatin 1 on the metabolism and cytotoxicity of 1-β-d-arabinofuranosylcytosine in human leukemia cells☆

Bryostatin 1 is a macrocyclic lactone protein kinase C (PK-C) activator which has demonstrated promising antileukemic activity in preclinical studies. We have examined the effect of this agent on the metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) in both log phase and high-density human promyelocytic leukemia cells (HL-60). Exposure of low-density cells to 12.5 nM bryostatin 1 for 24 hr prior to a 4-hr incubation with 1 or 10 microM ara-C resulted in nearly a 2-fold increase in ara-CTP formation. When cells were maintained under high-cell density conditions (e.g. 5 x 10(6) cells/mL) for 24 hr prior to ara-C exposure, a 90% reduction in ara-CTP formation and ara-C DNA incorporation was observed. However, coincubation of high-density cells with bryostatin 1 for 24 hr increased ara-CTP formation 6- to 8-fold, yielding levels essentially equivalent to those achieved in low-density cells. Smaller (but still significant) increases in ara-C DNA incorporation were also noted. Enhancement of ara-CTP formation by bryostatin 1 occurred over a broad ara-C concentration range (0.1 to 100 microM), involved a temperature-dependent process, could not be mimicked by addition of hematopoietic growth factors, and was not related to neutralization of toxic or inhibitory substances in high-density medium. Exposure of cells to bryostatin 1 did not lead to morphologic or functional evidence of HL-60 cell maturation or an increase in cell viability, but did produce a decline in cellular proliferative activity as determined by thymidine and bromodeoxyuridine incorporation and cytofluorometric analysis. Bryostatin 1 did not exert its effects in high-density cells by inhibiting ara-C deamination or by interfering with ara-CTP dephosphorylation, but instead appeared to act by enhancing ara-C phosphorylation. Although cell-free extracts obtained from high-density cells exposed to bryostatin 1 exhibited levels of deoxycytidine kinase activity compared to controls, treated cells did display a significant decline in intracellular dCTP levels (e.g. 0.7 vs 1.3 pmol/10(6)), and nearly a 2-fold increase in ATP and UTP concentrations. Ara-CTP formation was also increased substantially by other PK-C activators including phorbol dibutyrate and mezerein (10-100 nM); this process was inhibited more than 70% by the PK-C inhibitor H-7 (50 microM), but not by the PK-C inhibitors staurosporine, tamoxifen, and HA1004. Finally, coadministration of ara-C and bryostatin 1 resulted in greater than expected inhibitory effects toward HL-60 cell clonogenic growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Establishing Paternity Using Minisatellite DNA Probes When the Putative Father Is Unavailable for Testing

A paternity case involving a putative father who had died a few years earlier in an automobile accident was referred to the laboratory for testing. The child and his mother, the deceaseds parents, and nine of the deceaseds siblings were available for analysis. As previously reported, paternity testing using red blood cell groups, human leukocyte antigens (HLA), red blood cell enzymes, serum proteins, and immunoglobulin allotypes gave a cumulative paternity index of 43,300 and a combined probability of paternity equal to 99.998%. RFLP analysis using Hinf I and Sau 3A single digests and the minisatellite deoxyribonucleic acid (DNA) probes 15.1.11.4 and 6.3 showed no exclusion of paternity and gave nearly conclusive evidence that the putative father was the biological father of the child.

The Human onc Gene c-myc: Structure, Expression, and Amplification in the Human Promyelocytic Leukemia Cell Line HL-60

Substantial evidence indicates that retroviral transforming (v-onc) genes originated by means of recombination between a present nontransforming virus and normal cellular sequences (Duesberg et al., this volume). These sequences, called cellular onc gene, are highly conserved during evolution, suggesting that they may code for protein products which are essential for cell growth or tissue differentiation. As these normal cellular genes are homologous to viral- transforming genes, their potential role in tumorigenesis is of great interest. As an alternative to direct transformation by a viral onc gene, abnormal activation of a cellular onc gene may cause transformation. Two models have been proposed for such a mechanism. First, high levels of expression of a cellular onc gene may be caused by the insertion nearby of a viral promoter [12, 15, 16, 17] or by alteration of the physiological promoter by a mutagenic agent such as a chemical carcinogen. Secondly, a cellular onc gene may be relocated in a transcriptionally active region of the genome as a consequence of chromosomal rearrangements [2, 5, 6, 13]. In this chapter we review the evidence for a possible third mechanism for onc gene activation in neoplastic cells, that of gene amplification. The human homologue, c-myc, of the transforming gene of avian myelocytomatosis virus (MC29), which is expressed at relatively high levels in the human promyelocytic leukemia cell line HL-60, is stably amplified in the genome of these cells [7]. Amplification was also detected in primary, uncultured leukemic cells from the same individual, suggesting that the c-myc amplification may have been involved in the leukemic transformation in this case.

Use of phorbol-12,13-dibutyrate as a mitogen in the cytogenetic analysis of tumors with low mitotic indexes

Solid tumors, particularly those involving the colon, breast, and lung, are the most common tumors in humans. However, many technical difficulties exist in obtaining analyzable chromosomes from these tumors, including the inability to stimulate cell division. Phorbol-12,13-dibutyrate (PDBu) is a tumor promoter that activates a variety of cellular responses, including proliferation. Using flow cytometry, we have demonstrated that PDBu acts as a mitogen in primary cultures of colon tumor cells. Based on these results, we developed a short-term culture technique that greatly improves the yield of analyzable metaphases from colon tumors. Stimulated cultures consistently contained at least ten times more metaphases than unstimulated cultures, and chromosome morphology was improved. By modifying this technique with the addition of the calcium ionophore A23187, we have successfully obtained analyzable chromosomes from the peripheral blood of normal individuals, chronic lymphocytic leukemia patients, and a nodular small cell lymphoma patient. These results demonstrate that mitogenic stimulation by PDBu is a valuable technique in the cytogenetic analysis of colon tumors. By using PDBu alone or in combination with other agents, this technique may also be applicable to many other tumors that are difficult to karyotype because of an inability to obtain mitoses.

Characterization of a rearrangement in the c-MYB promoter in the acute lymphoblastic leukemia cell line CCRF-CEM

Despite the frequent description of 6q- structural abnormalities in human leukemias and lymphomas, rearrangements of the c-MYB locus have not been detected. We have detected a rearrangement in the c-MYB proto-oncogene in the cell line CCRF-CEM, an immature T-cell leukemia cell line which is not 6q-. Due to this rearrangement, a large portion of the c-MYB promoter conserved between the human and murine c-MYB genes is lost. The rearranged locus, which we have designated MRR (MYB rearranged region), has been cloned and mapped to chromosome 6. Field inversion gel electrophoresis (FIGE) studies reveal that the MRR sequence is linked to the c-MYB locus, suggesting that the rearrangement is due to a submicroscopic deletion. The rearrangement appears to have no effect on c-MYB promoter activity as analyzed in CCRF-CEM cells. The normal locus of the MRR sequence has been cloned from a human placental genomic library. Partial sequence analysis of this clone reveals that a portion of the DNA lost in the rearrangement shows a high degree of homology to a member of the myc family of oncogenes. Thus the characterization of this rearrangement has yielded a new set of probes for the study of chromosome 6q abnormalities in human leukemias and lymphomas and provides the first evidence for potential involvement of the c-MYB locus itself in submicroscopic deletions within chromosome 6.

Cellular onc Genes: Their Role as Progenitors of Viral onc Genes and Their Expression in Human Cells

Viral transforming (v-onc) genes are derived from cellular (c-onc) genes that are highly conserved among vertebrates. Comparative studies of v-onc and c-onc genes have shed some light on the mechanism leading to formation of the transforming viruses. A specific example of the sis gene is presented here for illustration. Studies on the expression of six c-onc genes in human cells revealed at least three categories of onc genes: (a) those that are universally expressed and probably are important in basic cellular functions, (b) those that are not detectably expressed in the cells examined and may have very transient expression in development, and (c) those that are only expressed in specific cell types and may be important in tissue differentiation. Our studies do not show conclusively a role of these onc genes in human neoplasias.

Retroviruses and Onc Genes in Human Leukemias and Lymphomas

Retroviruses are of special importance in the understanding of the origin and pathogenesis of leukemias and lymphomas of man. Not only are they a major cause of naturally occurring leukemias and lymphomas in several animal species, thus providing useful model systems relating to the cause and development of neoplasias in man, but they also provide a means to identify and study some cellular genes (so-called onc genes) which are important in cell growth and differentiation and which have been transduced by the acutely transforming retroviruses. Many people believe that abnormal expression and/or qualitative modification of the c-onc genes are involved in the molecular pathogenesis of most or even all cancers no matter what the cause.

Detection of a surface antigen on NIH3T3 cells transfected with a human leukemia oncogene

This study was conducted to examine the cell surface changes associated with oncogene induced transformation. Using the transfection technique the DNA of a human acute lymphocytic leukemia (ALL) was used to transform murine NIH3T3 cells. Balb/c mice were immunized with these transfectants and their immune splenocytes were used to produce a monoclonal antibody (17-9H3). Antibody 17-9H3 was demonstrated to bind to the cell membranes of transfectants and leukemia cells but not normal 3T3 cells in an enzme linked immunosorbent assay. Fresh human leukemias, cultured leukemia lines and normal hemopoietic cells were examined with immunoperoxidase staining techniques to determine the specificity of 17-9H3. Our data suggest that the antigen associated with a human acute lymphocytic leukemia oncogene is ubiquitous, distributed among both neoplastic and normal hemopoietic cells. Among fresh human leukemias the antigen appears to be present primarily on fresh null ALLs and chronic myelogenous leukemia (CML) in blast crisis. This antigen was also found to be expressed by the majority of cultured T-cell lines tested.

The Cloning and Analyses of Human Cellular Genes Homologous to Retroviral Onc Genes

Type-C retroviruses are associated with certain forms of naturally occurring leukemias and lymphomas of many species, including humans, as recently evidenced. These viruses generally do not transform cells directly in vitro and apparently do not contain a specific transforming gene. In contrast, a more unusual class of retroviruses is the acutely transforming viruses1,2. They cause diseases rapidly in vivo, have the capacity to transform appropriate target cells in vitro, and contain genomes which are usually defective for replication and include a specific transforming (v-onc) gene.