Michael J. Ahearn

Long-term culture of lymph node tissue from a patient with lymphocytic lymphoma. II. Preliminary ultrastructural, immunofluorescence and cytogenetic studies.

The original cell line was established from a lymph node biopsy specimen from a 59‐year‐old male patient having lymphocytic lymphoma (chronic lymphocytic leukemia). The culture has maintained an identical pattern of growth for 18 months. Two morphologically distinct cell types are present. One is a spindle shaped, flattened element attached to the glass surface, often forming an interlacing network; the other appears as clusters of ovoid or round floating elements. A first report described immunofluorescent studies with antihuman gammaglobulin, in which the round floating elements displayed marked fluorescence. This second report mentions similar results obtained using the patients labeled gammaglobulin. Preliminary ultrastructural studies revealed elementary particles resembling virus in the same cells. Chromosomal analysis of the strain showed a change to aneuploidy at approximately 6 months.

General implications of chromosomal alterations in human leukemia

Abstract Analysis of bone marrow cells is essential for the complete cytogenetic characterization of leukemia. The important technical requirements for dealing with bone marrow include anticoagulation, prompt processing, removal of excessive numbers of erythrocytes, inoculation of optimal numbers of cells, use of fetal calf serum, use of hypotonic phosphate buffer to swell the cels, and photography with a camera that produces a large negative. Seventy-five to 90 per cent of adult patients with chronic myelogenous leukemia have the Philadelphia (Ph 1 ) chromosome in bone marrow cells. Patients with the Ph 1 chromosome have a better prognosis than do those who lack it. chromosomal studies during the acute transformation (“blastic crisis”) of chronic myelogenous leukemia have shown a high incidence of abnormal karyotypes in which one or more Ph 1 chromosomes or other markers are present, suggesting that a process of clonal evolution has produced clones that are more resistant to therapy. In a series of 170 patients with acute leukemia who had sequential bone marrow cytogenetic studies, we have found the incidence of aneuploidy to be 40.5 per cent. Most of these patients had acute myelogenous leukemia. Fortysix per cent of the aneuploid karyotypes were hyperdiploid, 25 per cent were hypodiploid, and 29 per cent were pseudodiploid. Contrary to general opinion, certain of the aneuploid cytogenetic profiles had a tendency to occur in repetitive fashion in genetically unrelated patients. Certain profiles also carried prognostic implications. Patients with diploid and hyperdiploid karyotypes survived for 49 weeks (mean survival time), whereas those with pseudodiploidy and hypodiploidy survived for only 36 and 13 weeks, respectively. Our studies have shown repeatedly that a favorable response to therapy is marked by early disappearance of the aneuploid or pseudodiploid clone and that this same abnormal clone reappears when the leukemia relapses. By contrast, in patients with chronic myelogenous leukemia in remission, the Ph 1 chromosome persists in the bone marrow at its initial frequency. Electron microscopic studies on aliquots of the bone marrow samples examined for cytogenetic study have shown a high incidence of nuclear “blebs” in aneuploid leukemic bone marrows. These “blebs” appear to be derived from excessive proliferation of the nuclear membrane, disappear along with the aneuploid clone during remission, and reappear at the time of relapse. Cytogenetic analysis has provided the clinician with a useful new tool to characterize the leukemias.

Correlated Cytogenetic and Ultrastructural Studies in Acute Leukemia

We have previously reported the results of a combined cytogenetics and ultrastructural investigation on sequential bone marrows from adult acute leukemia patients before, during, and after chemotherapy [2]. These studies have established a correlation in the occurrence of abnormal cytogenetic clones and a high frequency of ultrastructural nuclear blebs in the bone marrow of approximately forty-one percent of the adult acute leukemia patients presenting at this Institution in active disease. Although both of these nuclear abnormalities have been reported in acute leukemia, their association in aneuploidy had previously been unrecognized [3,4,6,7], Our initial study has indicated that, in active disease, the acute leukemia patient with an aneuploid clone also demonstrates a high frequency of ultrastructural nuclear blebs in the immature cells of the myeloid series. Furthermore, the response to therapy in these patients is marked by a reduction in the number of aneuploid or pseudodiploid clones and in the presence of the nuclear blebs; conversely, impending relapse is indicated by reappearance of the same abnormal clone and nuclear structure. Since previously observed structures morphologically identical to the nuclear blebs observed in the cells of these acute leukemia patients have been attributed to high level drug effects of a specific category of DNA inhibiting agents, we have been prompted to examine the leukemia associated nuclear blebbed or aneuploid cell for evidence of deranged DNA metabolism [1, 8].

The effects of phenazinium and phenothiazine upon mitochondrial metabolism

Abstract The effect of the antitumor agent “phenazinium” (1,3-diamino-5-methyl phenaziniurn chloride) upon mitochondrial oxidations and oxidative phosphorylation has been investigated. Mitochondrial suspensions were prepared from mammalian liver and kidney. Phenazinium produced a significant degree of inhibition of mitochondrial oxidations with several substrates, and, in concentrations of 1 mM or above, induced partial uncoupling of oxidative phosphorylation. These effects are compared to those produced by phenothiazine, and differences are discussed. The compound has been shown to produce severe ultrastructural changes in renal tubule cells in vivo , and the significance of these changes is related to the observed toxicity of the drug.