Michael A. Baker

ProcessingO-glycan core 1, Galβ1-3GalNAcα-R. Specificities of core 2, UDP-GlcNAc: Galβ1-3GalNAc-R(GlcNAc to GalNAc) β6-N-acetylglucosaminyltransferase and CMP-sialic acid:Galβ1-3GalNAc-R α3-sialyltransferase

To elucidate control mechanisms ofO-glycan biosynthesis in leukemia and to develop biosynthetic inhibitors we have characterized core 2 UDP-GlcNAc:Galβ1-3GalNAc-R(GlcNAc to GalNAc) β6-N-acetylglucosaminyl-transferase (EC 2.4.1.102; core 2 β6-GlcNAc-T) and CMP-sialic acid: Galβ1-3GalNAc-R α3-sialyltransferase (EC 2.4.99.4; α3-SA-T), two enzymes that are significantly increased in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). We observed distinct tissue-specific kinetic differences for the core 2 β6-GlcNAc-T activity; core 2 β6-GlcNAc-T from mucin secreting tissue (named core 2 β6-GlcNAc-T M) is accompanied by activities that synthesize core 4 [GlcNAcβ1-6(GlcNAcβ1-3)GalNAc-R] and blood group I [GlcNAcβ1-6(GlcNAcβ1-3)Galβ-R] branches; core 2 β6-GlcNAc-T in leukemic cells (named core 2 β-GlcNAc-T L) is not accompanied by these two activities and has a more restricted specificity. Core 2 β6-GlcNAc-T M and L both have an absolute requirement for the 4- and 6-hydroxyls ofN-acetylgalactosamine and the 6-hydroxyl of galactose of the Galβ1-3GalNAcα-benzyl substrate but the recognition of other substituents of the sugar rings varies, depending on the tissue. α3-sialytransferase from human placenta and from AML cells also showed distinct specificity differences, although the enzymes from both tissues have an absolute requirement for the 3-hydroxyl of the galactose residue of Galβ1-3GalNAcα-Bn. Galβ1-3(6-deoxy)GalNAcα-Bn and 3-deoxy-Galβ1-3GalNAcα-Bn competitively inhibited core 2 β6-GlcNAc-T and α3-sialyltransferase activities, respectively.

Specificity of Heteroantisera to Human Acute Leukemia-Associated Antigens

Antisera have been raised to human leukemic blast cells from individual patients in mice rendered tolerant with cyclophosphamide to remission leukocytes from the same individual. 10 antisera were raised against acute myelogenous leukemia (AML) cells and 5 antisera were raised against acute lymphoblastic leukemia (ALL) cells. Antisera to AML cells were absorbed with ALL cells, and antisera to ALL cells were absorbed with AML cells. Unabsorbed and absorbed antisera as well as antisera raised in nontolerant mice were tested for cytotoxicity against various cells of a panel containing myeloblasts from 35 patients with AML, lymphoblasts from 7 patients with ALL, myeloblasts from 7 patients with chronic myelogenous leukemia (CML) in blast crisis, peripheral blood leukocytes from 12 patients with acute leukemia in remission and 30 nonleukemic patients, and nucleated bone marrow cells from 10 nonleukemic patients. Unabsorbed antisera to AML or ALL cells raised in tolerant mice were highly cytotoxic to leukemic blasts cells but significantly less cytotoxic to remission and control cells. Antisera to AML cells absorbed with ALL cells retained measurable cytotoxicity against AML cells but were not cytotoxic to ALL cells or control cells. Similarly, antisera to ALL cells absorbed with AML cells retained significant cytotoxicity only to ALL cells. Control antisera raised in nontolerant mice were cytotoxic to all cells tested. Although species specific, histocompatibility, differentiation, maturation, and cell cycle-associated antigens may be responsible in part for the cytotoxic activity of the unabsorbed antisera, the absorbed antisera are probably detecting antigens specific for their leukemic cell type.

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R beta 1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues.

UDP-GlcNAc:GlcNAc β1-2Manα1-6R (GlcNAc to Man) β1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAcβ1-6 branch to bi- and triantennaryN-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAcβ1-2Manα1-6Glc/Manβ-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Manα1-3 residue and the 4-hydroxyl of the Manβ- residue of the Manα1-6(Manα1-3)Manβ-RN-glycan core are not essential for catalysis but influence substrate binding. GlcNAcβ1-2(4,6-di-O-methyl-)Manα1-6Glcβ-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.

Malignant lymphoma of mucosa-associated lymphoid tissue of the lacrimal gland: case report and review of literature.

Mucosa-associated lymphoid tissue (MALT) lymphomas are increasingly recognized as a distinct clinical–pathologic entity among the non-Hodgkin’s lymphomas. It usually presents as a localized disease process in extranodal tissues or organs such as stomach, salivary gland, thyroid gland, and not infrequently in orbital adnexa. Radiotherapy has an important role in the management, although long-term clinical results specifically addressing localized MALT lymphomas are lacking. We report a case of localized MALT lymphoma of the lacrimal gland, with successful treatment by radiation therapy (total dose 25 Gy) with 3 years of clinical follow-up. A review of the published literature was undertaken to assess the role of radiotherapy in the treatment of this disease involving orbital tissues, specifically, the lacrimal gland. Based on previous reports of patients with orbital lymphomas (low grade) and pseudolymphomas, of which many will now be recognized as MALT lymphomas, radiotherapy has an excellent local control rate and would be the treatment of choice. However, long-term results of pathologically confirmed cases of MALT lymphomas need further study because occasional relapses at distant sites can occur.

Results of Therapy for Acute Myeloid Leukemia in First Relapse

Fifty-four consecutive patients with acute myeloid leukemia (AML) in first relapse presenting at a single institution were studied to determine factors affecting response to re-induction therapy. For purposes of analysis, re-treatment protocols were grouped into two categories, one with high dose and the other with standard dose cytosine arabinoside. Most regimens also included an anthracycline, mitoxantrone or amsacrine. Thirty-one of the 51 patients (61%) who received therapy achieved a second complete remission (CR-2). Median duration of CR-2 was 6 months (range 1-24+ months). Five patients remain in CR-2, three of whom received bone marrow transplants (median follow-up 24 months). The variables, age, gender, FAB subtype, leukocyte and platelet count, duration of CR-1, the initial and re-induction regimens were analyzed for prognostic value in attaining and maintaining CR-2. Only younger age (p < 0.001) and longer CR-1 duration (p < 0.05) were significantly correlated with greater likelihood of attaining CR-2 with univariate analysis, and only age was correlated with CR-2 rate using multivariate analysis (p = 0.018). Younger age was associated with longer CR-2 duration (p = 0.003) using multivariate analysis, a correlation that persisted when transplanted patients were excluded. There was no advantage to the use of high dose versus standard dose cytosine arabinoside in the reinduction regimen with respect to the ability to either achieve or sustain CR-2. Our data indicate that although the remission induction rate for AML in first relapse is high, remissions are brief and other strategies are required to improve outcome of patients in second remission.

Activated Protein Kinase C Directly Phosphorylates the CD34 Antigen in Acute Lymphoblastic Leukemia Cells

The precursors of all blood cell lineages are contained within the 1-3% of bone marrow cells which express the CD34 antigen, and this population can reconstitute the hematopoietic system of lethally irradiated animals and humans. A potential regulatory role for the CD34 antigen in progenitor cell function and differentiation was indicated by our recent findings that the CD34 antigen can be phosphorylated in vivo to high stoichiometry in primitive CD34+ cell-lines by activated protein kinase C. To exclude the possibility that these effects were restricted to cell-lines, we have performed similar experiments on fresh cells from a patient with drug-resistant acute lymphoblastic leukemia. Similar to our previous findings, we found the CD34 antigen to be hyperphosphorylated in lymphoblasts labeled in the presence of active phorbols. The same peptides which were hyperphosphorylated in phorbol-stimulated cell-lines were also phosphorylated in phorbol-stimulated lymphoblasts. These data indicate that CD34 is a substrate molecule for PKC in fresh CD34+ lymphoblasts and underline the role of modulators of PKC activity in the biology of primitive leucocytes.

Extensive Cutaneous Plasmacytomas

An unusual case of extensive cutaneous and subcutaneous plasmacytomas is reported. Breast involvement was also a feature in this individual. The relevant literature relating to this unusual presentation of myeloma is reviewed.

Membrane glycoprotein changes associated with anthracycline resistance in HL-60 cells.

SummaryThe glycoproteins on the surface of HL-60/S wild-type, drug-sensitive human leukemia cells and HL-60/AR anthracycline-resistant cells which do not overexpress the P-glycoprotein, were characterized by labeling with [35S]-methionine, NaB[3H4], phosphorus 32, or sodium iodide I 125. HL-60/S and HL-60/AR cell lysates and membrane fractions tagged with [35S]-methionine or phosphorus 32 showed no significant differences in their protein patterns as analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and by autoradiography. HL-60/S cells labeled with NaB[3H4] yielded glycoproteins that were smeared predominantly in the molecular-weight range of 210,000 and 160,000 Da, with pI values ranging between pH 4 and pH 4.4. In contrast, NaB[3H4]-labeled HL-60/AR cells showed 7–8 discrete glycoproteins within a molecular-weight range of 170,000 and 140,000 Da, with pI values also ranging between pH 4 and pH 4.4. In addition, [3H]-glucosamine incorporation into HL-60/S and HL-60/AR cells revealed that the latter showed lower uptake of [3H]-glucosamine than did the former. Following treatment with tunicamycin, [3H]-glucosamine uptake in HL-60/S cells decreased, whereas that in HL-60/AR cells remained unchanged. Surface-membrane radioiodination of HL-60/S and HL-60/AR cells showed two distinct protein electrophoretic patterns, with differences being observed in both the high-(220–95 kDa) and low-molecular-weight ranges (21 kDa). Flow cytometric analysis of HL-60/S and HL-60/AR cells using myeloid and lymphoid antigen-specific antibodies demonstrated no antigenic differences between HL-60/S and HL-60/AR cells. HL-60/S cells incubated in the presence of tunicamycin, an inhibitor ofN-linked glycosylation, or the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) developed a glycoprotein pattern similar to that observed in HL-60/AR cells. In addition, tunicamycin treatment of HL-60/S cells decreased daunorubicin (DNR) retention and altered its intracellular distribution as compared with that in HL-60/AR cells. These data indicate that HL-60/AR cells do not possess either de novo or amplified high-molecular-weight surface-membrane proteins; instead, existing proteins are hypoglycosylated. These results also show that HL-60/AR cells exhibit the multidrug-resistant phenotype in association with altered membrane glycoproteins of both high (220–95 kDa) and low molecular weight (21 kDa), but without overexpression of the P-glycoprotein. Furthermore, in HL-60/S cells, the multidrug-resistant phenotype is partially inducible by inhibition ofN-linked glycosylation of cell-surfac proteins.

Changes in the granulocyte membrane following chemotherapy for chronic myelogenous leukaemia

Summary. Granulocytes from patients with chronic myelogenous leukaemia (CML) have been previously shown to have aberrant sialylation of membrane glyoproteins. We have examined the granulocytes from CML patients receiving intermittent chemotherapy to determine the relationship of the oligosaccharide changes to treatment. Compared to cells from non‐leukaemic patients, granulocytes from untreated CML patients showed less adherence to nylon wool, decreased reactivity with peanut lectin, and decreased binding of the synthetic chemotactic peptide formyl‐methionine‐leucine‐phenylalanine (FMLP). Granulocytes from CML patients treated with chemotherapy showed nylon wool adherence, peanut lectin reactivity and FMLP binding comparable to non‐leukaemic cells. Chemotherapeutic agents may interfere with oligosaccharide synthesis with a resulting change in the composition of cell surface glycoconjugates.

Adult Acute Lymphoblastic Leukaemia: The Value of Therapy Intensification

Results of multiagent chemotherapy protocols as therapy for adult acute lymphoblastic leukaemia in non-randomised multi-institutional studies appear superior to historical controls of less aggressive treatment. However, randomised studies have produced conflicting data and increasing the dosage of chemotherapy in conjunction with bone marrow transplantation has been disappointing. We reviewed retrospectively our experience with 68 adult ALL patients treated over a ten year period. Twenty-six patients received a standard four drug induction regimen without intensification. A second group of thirty-four patients received induction, intensification and re-induction phases of treatment. The results suggest that intensification of therapy does not alter median survival or median duration of remission, although a trend towards more long term disease-free survivors is observed.