Mitochondrial Electron Transport Enzymes in Normal and Leukemic Human Leukocytes

Assays of TD transhydrogenase and succinate cytochrome C reductase carried out on the homogenate of normal and leukemic human leukocytes andon the 1.5 M. fraction (fraction 3) of a continuous sucrose density gradientanalysis of a crude mitochondrial fraction, provided strong evidence for themitochondrial location of TD transhydrogenase. A slightly higher activity ofthese enzymes was found in the homogenate of cells from chronic lymphocyticand acute leukemia compared to those of normal polymorphonuclear leukocytes, normal lymphocytes, and cells from chronic myeloid leukemia. Thesedifferences were very much more marked when purified mitochondrial fractions from these cells were examined. The significance of these findings is discussed.

Submitted on August 9, 1967 Accepted on December 6, 1967     

The RNA Nucleotide Composition in Human Leukocytes from Normal and Leukemic Cases

The RNA-nucleotide composition of leukocytes from normal and leukemicpatients has been analyzed. The amount of the four mononucleotides per 10⁹leukocytes is significantly increased in patients with either chronic lymphocyticor chronic granulocytic leukemia when compared with leukocytes from normalsubjects. High values have been observed in acute granulocytic leukemia andduring blastic crisis occurring in cases of chronic granulocytic leukemia. Thissuggests that the mononucleotide increase may be correlated with the numberof blast cells. The molar base composition of the 4 mononucleotides and theratio of 6-keto/6-amino acids and that of purine/pyrimidine bases in all leukemic cells examined were similar to the ratios occurring in normal leukocytes.

The significance of these findings, relative to the theory of viral origin ofhuman leukemia, is discussed.

Submitted on December 27, 1965 Accepted on May 22, 1967     

Mucopolysaccharide Sulfation in Normal and Leukemic Leukocytes

Mucopolysaccharide sulfation wasdemonstrated in leukemic granulocyticprecursors in short-term suspensionculture by incorporation of ³⁵SO₄ intoa compound identified chromatographically as chondroitin sulfate. Thegel filtration pattern of sulfated mucopolysaccharide obtained from leukemic leukocytes was qualitatively similar to that found with normal granulocytic precursors. Sulfation of mucopolysaccharide was about 50% of thenormal level in cells from chronicgranulocytic leukemia, and approximately 15% of the normal level incells from acute granulocytic leukemia or chronic granulocytic leukemiain blastic crisis. Lymphocytes fromacute and chronic lymphocytic leukemia showed only traces of sulfate incorporation. Leukocyte mucopolysaccharide sulfation was studiedin cultured cells from patients withacute leukemia to determine whetherthis reaction could aid in distinguishing acute granulocytic leukemia fromacute lymphocytic leukemia. Significant levels of sulfation were obtained in cells from all leukemiasjudged to be acute granulocytic,while virtually no incorporation wasfound in cells from acute lymphocytic leukemia when a 24-hr incubationperiod was employed. In studies employing a 3-hr incubation, agreementas to the cell of origin of the leukemiaand the degree of sulfation in the intracellular fraction was obtained in 16of 18 determinations performed on 16patients with acute leukemia. It isproposed that determination of ³⁵SO₄incorporation in the intracellular fraction may help to differentiate acutegranulocytic and acute lymphocyticleukemia.

Submitted on October 8, 1971 Revised on June 8, 1972 Accepted on June 9, 1972     

Nuclear DNA Sequences Present in Human Leukemic Cells and Absent in Normal Leukocytes

The central purpose of the present study was to test the proposition that the nuclear DNA of every human cell contains whatever information is necessary and sufficient for transformation to malignancy. The experiments were made possible by our earlier identification in human leukemic cells of particulate elements encapsulating 70S RNA and RNA-directed DNA polymerase. The [(3)H]DNA synthesized by these particles was used as a probe, through molecular hybridization, to normal and leukemic DNA. The results obtained establish that leukemic nuclear DNA contains particle-related sequences that cannot be detected in normal leukocytes. This outcome does not support the virogene-oncogene theory, which postulates the inclusion of at least one complete copy of oncogenic information in the genome of every normal cell.The data suggest that we may not be forced to cope with an omnipresent DNA segment coding for malignancy. Under the circumstances, we can perhaps entertain more hopeful pathways leading to the control and cure of cancer.     

Lactic Dehydrogenase of Human Chronic Lymphocytic Leukemic Leukocytes

Lactic dehydrogenase was quantitated and its isozyme pattern studied inthe leukocytes of 10 patients with chronic lymphocytic leukemia. There wasa wide range of LDH activity, although all isozyme patterns by starch-gelelectrophoresis showed greatest activity in the No. III isozyme band. Comparison of the rate of migration of the leukemic leukocyte LDH with LDHfrom normal leukocytes and erythrocytes showed no difference in migrationrate, suggesting that the LDH of chronic lymphocytic leukemic leukocytesdoes not differ in electrical charge from LDH of normal leukocytes.

Submitted on March 4, 1965 Accepted on May 19, 1965     

RNA Metabolism of Normal and Leukemic Leukocytes: II. Ribonuclease

Ribonuclease has been partially purified from human leukocytes and itsproperties investigated. The enzyme has a pH optimum between 6 and 6.5.It is equally active with RNA prepared from various sources, attacking phosphodiester bonds adjacent to pyrimidine bases. Activity in myeloblasts orlymphocytes is about one-tenth that of mature granulocytes.

Submitted on March 14, 1966 Accepted on May 10, 1966     

Colony Formation by Normal and Leukemic Human Marrow Cells in Culture: Effect of Conditioned Medium From Human Leukocytes

"Conditioned medium" obtained fromcultures of human peripheral leukocytespromoted the growth of human marrowcells in cell culture. This material alsopermitted the growth of small coloniesfrom the marrow of patients with acutemyelogenous leukemia in relapse; in itsabsence, only occasional colonies wereobserved.

Submitted on June 30, 1970 Accepted on July 24, 1970     

Deoxycytidine Pathway in Separated Normal and Leukemic Leukocytes With Effects of Cell Culture

Enzymes of dC metabolism were studiedin glass-column-separated normal andleukemic cells. Supernatants of sonicatedcells were incubated with dC-³H ordCMP-³H and the end products separated with paper chromatography. Greatest recovery of dCTP, dUMP, and TTPoccurred with myeloblasts and monoblasts. Mature granulocytes, and normaland leukemic lymphocytes gave the greatest breakdown to uracil. Myelocytes andlymphoblasts gave intermediate results.Leukemic lymphocytes from different patients varied greatly in their end products, but most did show evidence ofTMP synthetase activity contrary toprevious reports. Recovery of products ofTMP synthetase and, to a lesser extent,dCMP deaminase and dC kinase activities was greatest in cells having or developing a potential for mitosis. CLL-Lwhose extracts showed appreciable production of dCTP, dUMP, and TdR compounds, however, did not regularly produce blast cells in response to PHA inculture. Cellular extracts of N-L, orCLL-L from cases which responded toPHA did show, on the other hand, aconsiderable increase in accumulation ofthese compounds from initial low valuesduring culture.

Submitted on February 5, 1971 Revised on April 12, 1971 Accepted on April 28, 1971     

DNA Polymerase and Carbohydrate Metabolizing Enzyme Content of Normal and Leukemic Glass Column Separated Leukocytes

1. Enzymes of normal and leukemic glass column separated leukocytes wereassayed with fluorometric (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactic dehydrogenase, glyceraldehyde-3-phosphatedehydrogenase and isocitric dehydrogenase) and radioisotope (DNA polymerase) micromethods.

2. Most results were obtained by direct assay of the specific cell type. Somerequired simple calculation. It was demonstrated that the enzyme activityof mixed cell suspensions was the sum of the enzyme content of the componentcells, at least for the enzymes studied.

3. Assays of mixed cell suspensions may give an erroneous picture. Thuschanges in differential cell counts, as well as alterations in enzyme content ofindividual cells, must be taken into account for correct interpretation.

4. Changes in enzyme content of mixed cell suspensions after therapy weredue chiefly to alterations in the differential cell count rather than to changesin the enzyme content of individual cell types.

5. The patterns of assays of the carbohydrate metabolizing enzymes studied,except isocitric dehydrogenase, paralleled each other. Highest values werefound in mature PMN leukocytes and lowest in blasts. With isocitric dehydrogenase absolute values were much lower, while the highest assays werefound in myelocytes.

6. DNA polymerase content correlated well with the ability of a cell todivide. It was highest in blasts and lowest in mature PMN leukocytes.

Submitted on May 27, 1965 Accepted on August 6, 1965     

Malic and Lactic Dehydrogenase Isozymes of Normal and Leukemic Leukocytes Separated on Glass Bead Columns

Five LDH and two MDH isozyme bands were obtained with acrylamidegel electrophoresis of leukocyte extracts. Normal lymphocytes showed a hightotal H-LDH (heart type) activity (67 per cent) with 25 per cent in LDH-1and only 3.5 per cent in LDH-5. Lymphocytes from chronic lymphatic leukemia(CLL) and lymphosarcoma leukemia (LSA-LL) had less LDH-1, and moreLDH-3 and LDH-4, than normal lymphocytes. The H-LDH fell to 60.5 per centin CLL and 56 per cent in LSA-LL. PMN leukocytes had low H-LDH activity(38.8 per cent) with 3.3 per cent in LDH-1 and 25.8 per cent in LDH-5. Inmyelogenous leukemia, myeloblasts had the most LDH-1 and H-LDH, whilemature PMN had the least. PMN leukocytes isolated from CLL, LSA-LL, andmyelogenous leukemia had LDH patterns like the normal. Monocytes fromacute monocytic leukemia were low in LDH-1 and LDH-5, but had a high totalenzyme content. They evidently were rich in LDH-2, 3, and 4.

Lymphocytes had less MDH-1 (60 per cent) than PMN leukocytes (78 percent). In CLL, lymphocyte MDH-2 increased. In myelogenous leukemia,myeloblasts had the most MDH-2 and mature PMN the least. Monocytesfrom monocytic leukemia contained a little more MDH-2 than PMN leukocytes.

In general, white cell immaturity and/or ability to divide was associatedwith high levels of LDH-1, total H-LDH, and MDH-2.

Submitted on May 5, 1966 Accepted on June 26, 1966     

DNA Polymerases in Normal and Leukemic Human Hematopoietic Cells

DNA polymerase activities were assayedin bone marrow cells and peripheral leukocytes from normal people and patientswith acute myelogenous, chronic lymphocytic, and chronic myelogenous leukemia.Extracts of subcellular components werefractionated by velocity sedimentationthrough sucrose density gradients andassayed using activated DNA as template.Two major DNA-dependent DNA polymerases were found in human cells withmolecular weights of approximately50,000 and 200,000 daltons, respectively.The DNA polymerase of high molecularweight is located in the soluble cytoplasmic fraction and is inhibited byN-ethylmaleimide. The low molecularweight polymerase is detected in extractsof nuclei and in the soluble fraction. It isresistant to inhibition by N-ethylmaleimide. In all cell types tested, total DNApolymerase activities were much higher incytoplasmic than in nuclear extracts.Lymphocytes purified from normal peripheral blood had three to four times asmuch of both the high and low molecularweight polymerase activities per cell aspurified granulocytes. Leukemic myeloblasts had 10 to 20 times as much cytoplasmic DNA polymerase activity as moremature leukocytes from normal peripheralblood. In general, immature granulopoietic cells contained higher total DNA polymerase activities than more maturegranulocytes, and the major increases inpolymerase activities were in the highand low molecular weight cytoplasmicenzymes rather than in the nuclearenzyme.

Submitted on October 9, 1973 Revised on January 23, 1974 Accepted on January 28, 1974     

Ribonucleic Acid Biosynthesis in Human Leukocytes: The Fate of Rapidly Labeled RNA in Normal and Abnormal Leukocytes

Ribonucleic acid (RNA) was isolated from a variety of human leukocytepopulations exposed to tritiated uridine in vitro. Several species of RNA wereextractable from leukocytes in a phenol-water system. With mild conditions ofextraction a pH 7.6 fraction was obtained, which contained 60 to 75 per cent oftotal cellular RNA. After removal of this RNA component reextraction atelevated temperature and pH yielded a pH 9 fraction which contained 25 to 40per cent of total cellular RNA. The pH 7.6 fraction contained 28 and 18 Sribosomal RNA and 4 S RNA. The pH 9 RNA fraction was heterogeneouslydistributed in a sucrose density gradient. The 28 and 18 S components wereslowly labeled by H³-uridine and were relatively stable. The 4 S componentwas rapidly labeled and was unstable. The pH 9 RNA fraction contained mostof the rapidly labeled RNA, which was either of high molecular weight orheterogeneous as to molecular size.

The pattern of incorporation of H³-uridine into the various components ofRNA was similar in normal and neoplastic granulocytes and lymphocytes.

Rapidly labeled leukocyte RNA had the following characteristics: (1) itssynthesis was actinomycin sensitive; (2) it was unstable, having a half-life inthe range of 16 to 225 minutes; and (3) it was in part a precursor of ribosomalRNA.

The sensitivity of granulocyte protein synthesis to inhibition of RNA synthesis suggests that granulocytes have both stable and unstable templates.

Submitted on January 17, 1966 Accepted on March 26, 1966     

Histochemical Demonstration of Diaphorases and Dehydrogenases in Normal Human Leukocytes and Platelets

A method for the cytochemical demonstration of oxidative enzyme activityin the white cells and platelets of peripheral blood is described. The technicdepends upon the reduction of a colorless ditetrazolium salt to a blue granularinsoluble diformazan at the site of enzyme activity. Identification of the cellsis accomplished by phase microscopy thus avoiding the deleterious effects offixation and counterstaining. The distribution of oxidative enzyme activityin normal peripheral blood as observed by this method is discussed.

Submitted on December 5, 1960 Accepted on January 5, 1961     

Differentiation of Human Leukemic from Normal Lymphocytes by Limulns Serum Agglutination

Human peripheral chronic lymphocytic leukemic lymphocytes and a line (B411-4) of cultured human B cells are agglutinated by Limulus serum to a significantly higher titer and score than peripheral normal human lymphocytes or a line (MOLT-4-F) of cultured human T cells.