Biomedical subjects
Publications and source records attributed to I A Cooper.
Two patients with an initial diagnosis of a poorly differentiated lymphocytic lymphoma suffered a pathological rupture of the spleen in association with the histological transformation of the disease to a large cell lymphoma and the development of a leukaemic phase. The value of computerized tomographic scanning of the upper abdomen in confirming the diagnosis of splenic rupture is demonstrated.
SCC30 cells (derived from a single cell from the Chinese hamster ovary CHO-K1 cell line, selected on the basis of a stable chromosome complement) were used to select cell variants with hypomethylated DNA. Cells were treated with 5-aza-2'-deoxycytidine (5azadCyd) at 0.1, 1, or 5 microM for two weeks with the medium and drug renewed twice weekly. From the few surviving cells, 25 random single cell-derived clones were grown for freezing cell stocks, and for DNA isolation for 5-methyldeoxycytidine (5medCyd) estimations. After a minimum of one month's recovery from the drug, these cells showed a continuum of 5medCyd levels ranging from ones with the same as the parental clone (2.93%) to ones having lost almost 50% of their DNA methylation. The modal value corresponded to a loss of one third to one quarter of methylated sites. Five subclones with hypomethylated DNA were grown from the frozen stocks. These cells were shown not to be 5azaCyd-resistant cell variants. By the time sufficient cells had been grown to determine DNA methylation levels, the average percentage of 5medCyd had increased to 76% of the SCC30 value compared to 67% at the time of freezing cell stocks. However, this level of DNA hypomethylation remained constant over two months of continuous culture. Cells of one of these hypomethylated subclones were subjected to a second cycle of 5azaCyd treatment. Six random clones from the survivors showed a further decrease averaging 11% in the level of DNA methylation but, by two months in continuous culture, 5medCyd levels had returned to that present before the second cycle of selection. Hence, cell variants can be readily obtained which have lost some 8-10 million methylated sites (pairs of methylated deoxycytidines), and this loss does not compromise cell viability in in vitro culture. This is consistent with mammalian genomes containing a high level of background methylation in non-essential sites. The usefulness of such single cell-derived clones with stably hypomethylated genomes is discussed in relation to understanding the functions of deoxycytidine methylation in mammalian DNA.
In the majority of sites of methylation in the DNA of mammalian cells, the symmetry of methylation is restored within a few minutes of the passage of a replication fork. However, it has been shown that daughter strand methylation in immortalised cell lines is delayed in a substantial minority of sites for up to several hours after replication. We report here the results of two new approaches to the determination of the functional significance of delayed DNA methylation in mammalian cells. Firstly, we demonstrate that normal, nontransformed cells (human peripheral lymphocytes in short-term primary culture) have comparable proportions of delayed DNA methylation to many immortalised cell lines, showing that delayed DNA methylation is not just a secondary consequence of abnormally high methionine requirements commonly observed in transformed cells and that delayed DNA methylation would be unlikely not to occur in vivo. Secondly, we have used 5-aza-2'-deoxycytidine (5azadCyd) to derive subclones of cells from the Chinese hamster ovary cell line which have stably hypomethylated DNA. In three of these subclones which had lost on average one fourth of the methylation sites from their genomes, the proportion of daughter strand methylation which was delayed after replication was reduced by less than 10%. If delayed DNA methylation were site-specific, this implies that of the order of twice the number of "immediate" methylation sites than delayed methylation sites had been lost from the genomes of these hypomethylated subclones. Thus, delayed DNA methylation is an integral part of the process whereby replicating mammalian cells maintain the pattern of methylation in their genomes. These observations are discussed in relation to the significance of delayed DNA methylation for the accurate maintenance of methylation patterns in the genome and the consequent implications for the possible role of methylated deoxycytidines in mammalian gene control.
Flow cytometry studies for cellular DNA analysis were performed in 115 cases of non-Hodgkin's lymphoma, 53 of which had not received any prior chemotherapy or radiotherapy. DNA content was measured in ethanol fixed cells stained with chromomycin A3. According to the criteria of the International Working Formulation there were 43 low grade, 58 intermediate grade, and eight high grade lymphomas; six cases were in the miscellaneous group. Seventy seven (67%) had only diploid DNA content. Thirty eight (33%) showed DNA aneuploidy; 20 of these had been previously treated with chemotherapy or radiotherapy, or both. DNA aneuploidy was seen as hyperdiploidy in all cases except one, and it varied from slightly hyperdiploid to tetraploid. The incidence of aneuploidy increased significantly with increasing histological grade (p = 0.0002) and was not related to previous treatment. The low, intermediate, and high grade lymphomas had 14% (six of 43), 47% (27 of 58), and 62.5% (five of eight) cases, respectively, that showed DNA aneuploidy. The percentage of cells in S phase increased significantly with a higher histological grade (p less than 0.0001). The median S fraction in the low, intermediate, and high grade lymphomas was 1.0 (0.5 to 10)% 4 (0.4 to 35)%, and 27 (4.6-56)%, respectively. There is a significant correlation between histological grade and S fraction and the presence or absence of aneuploidy. There is heterogeneity, however, within both histological grade and a histological subtype.
Four types of plastic blood collection packs were studied for their ability to preserve platelet function during a 5 d storage period. The platelet concentrates were stored in polyvinyl chloride (Tuta Laboratories), PL 1240 and PL 732 (Fenwal Laboratories) and CLX (Cutter Laboratories) packs, on a Fenwal elliptical rotator at 20 degrees-24 degrees C. Plasma pH, lactate concentration, hypotonic shock response (HSR), platelet aggregation in response to ADP, collagen and ristocetin and levels of the plasticisers, di-2-ethylhexyl phthalate (DEHP) and tri-ethylhexyl trimellitate (TEHTM), were measured. Morphological changes were assessed by electron microscopy. No significant fall in pH occurred in any type of pack but in vitro function and platelet morphology was generally better preserved in Tuta and CLX packs than in PL 732 and PL 1240. Very little TEHTM leached out of the PL 1240 and CLX packs whereas the mean concentration of DEHP in the platelet concentrates stored in Tuta packs was 27.4 mg/100 ml plasma after 5 d of storage. The results indicate that it is possible to prepare and store platelet concentrates in polyvinyl chloride plastic packs for a period of 5 d and maintain their function and viability.
A 36 year old woman presented with a nasopharyngeal tumour which was diagnosed and treated as diffuse large cell lymphoma. Twelve mth later the patient developed acute myeloid leukemia. At this stage, the original biopsies were reviewed and considered in retrospect to be granulocytic sarcoma on the basis of staining for chloracetate esterase and lysozyme. She achieved and maintained marrow and peripheral blood remission with chemotherapy, but developed several cutaneous nodules and 2 breast lumps. One breast lump was excised and was found, by the use of monoclonal antibodies, to carry myeloid markers. Thus monoclonal antibodies provided additional confirmatory evidence for the diagnosis of granulocytic sarcoma.
The clinical, pathological and immunological features of a case of mantle zone lymphoma are described. The patient presented at the age of 16 with a history of painless enlargement of the inguinal lymph nodes, biopsy of which revealed a nodular small cell lymphoma. During the course of 11 yr he was treated with total nodal irradiation, splenectomy and combination chemotherapy at different times. A recent lymph node biopsy reviewed along with the previous node biopsies was diagnosed as mantle zone lymphoma. At this stage, the immunological studies showed that the neoplastic lymphoid cells had characteristic markers of mantle zone lymphocytes. He is asymptomatic with mild generalized lymphadenopathy 11 yr after the initial diagnosis. This case illustrates the diagnostic and therapeutic problems which may be encountered. Detailed immunological marker studies with an extended panel of monoclonal antibodies are described.
A high incidence of severe peripheral neuropathy occurred during the pilot study of a new regimen for the treatment of non-Hodgkin's lymphoma. The clinically observed incidence and severity of vincristine-induced peripheral neuropathy was considerably enhanced by the sequential use of vincristine and teniposide in this combination chemotherapy.
The case of a patient with Charcot-Marie-Tooth syndrome and diffuse large-cell lymphoma, in whom a severe generalized weakness developed after the intravenous administration of vincristine (2 mg) during combination chemotherapy, is reported. Spontaneous resolution of the severe weakness occurred when teniposide was substituted for vincristine in the chemotherapy regimen.
We describe the case of a 53-year-old woman in whom early stage large cell lymphoma appears to be associated with secondary erythrocytosis. A haemogram obtained six months earlier had been normal, but at the time lymphoma was diagnosed, investigations showed haemoglobin 20.5 gm/dl, total red cell count 6 X 10(12)/liter, haematocrit 0.60 liters/liter, whole blood volume 81.1 ml/kg, red cell volume 43.2 ml/kg, and plasma volume 37.8 ml/kg. Other investigations revealed no evidence of any other myeloproliferative disorder, and common causes of tissue hypoxia and other well-known causes of secondary polycythaemia were excluded. Combination chemotherapy employed to treat lymphoma reverted the abnormal blood counts to normal. We believe this to be the first described case of an association of lymphoma and secondary polycythaemia.
In 44 out of 758 patients (5.8%) with non-Hodgkin's lymphoma presenting between 1971 and 1982, the central nervous system (CNS) was involved. Patients with a diffuse histology had a 7.6% (34/449) incidence of CNS involvement compared to 3.9% (10/257) for patients with nodular lymphoma. In 63% of patients there was evidence of progressive systemic lymphoma at the time of diagnosis of CNS disease and in 23% CNS relapse occurred in clinical remission. Bone marrow was involved in 34% of patients at diagnosis and in 52% at some time prior to the onset of CNS complications. Cerebrospinal fluid cytology was positive in 63% and an elevated protein level was found in 95% of patients. The median length of survival of the 44 patients was only 3.2 months, but patients who responded to treatment of CNS lymphoma survived significantly longer than those who showed no response or progressed on therapy. Complete response to CNS treatment was achieved in five patients, of whom none relapsed in the CNS and two are long-term disease-free survivors. CNS prophylaxis appears justified for patients with lymphoblastic lymphoma, Burkitt's tumour, and diffuse undifferentiated lymphoma, who are at high risk of developing CNS complications. Patients with diffuse histiocytic, and diffuse poorly differentiated lymphocytic, lymphoma who have bone marrow involvement may also benefit from CNS prophylaxis.
Cells of the Chinese hamster ovary cell line were used to study the process of cell death induced by pulse treatment with 1-beta-D-arabinofuranosylcytosine (ara-C). Cells were synchronized by mitotic selection and pulse treated in early S phase with a concentration of ara-C (1 mM) which was sufficient to reduce plating efficiency to a few percentages of the control. The process of when and how the lethally damaged cells die was studied using a series of techniques in parallel. These included time-lapse microcinematography, flow microfluorimetry, and chromosome morphology in both anaphases/telophases and Colcemid-arrested metaphases. Most of the lethally damaged Chinese hamster ovary cells progressed through one, and many through two, cell cycles before death occurred. The cell death and abnormal divisions can be accounted for by the chromosome aberrations observed in Colcemid metaphases and anaphases/telophases. Death without any attempted division occurred between 3 and 9 normal cell cycle times after ara-C treatment. Chinese hamster ovary cells were also treated continuously with 1 mM ara-C. Under these conditions, cell death was still primarily division related. We argue that these data are not consistent with the actual incorporation of ara-C moieties into DNA being the primary cause of cell death. The data are discussed in relation to the postulated molecular mechanisms of toxicity of this drug.
We have determined the DNA renaturation kinetics for those DNA sequences of the Chinese hamster ovary (CHO-K1) cells in which enzymatic cytosine methylation occurred immediately after strand synthesis and for those in which methylation was delayed after strand synthesis. DNA sequences showing immediate or delayed methylation were found to be distributed throughout all repetition classes of the DNA of these cells, with a slight concentration of immediate methylation in moderately repetitive sequences and with delayed methylation being slightly over-represented in the highly repetitive fraction. However, DNA sequences showing both classes of methylation were represented equally in unique DNA sequences. We interpret these data to mean that the methylase acting near the replication forks (the 'immediate' methylase) is a relatively inefficient enzyme, missing some 20% of hemimethylated sites produced by DNA replication in these cells. We suggest that the methylase performing maintenance methylation at sites remote from the replication forks (the 'delayed' methylase) is simply a back-up enzyme for the first and that it has no true sequence specificity. The implications of this for the function(s) of DNA methylation in mammalian cells are discussed.
The effects of retinoic acid on the clonal growth in nutrient agar of neutrophil and macrophage progenitors from normal bone marrow and fetal liver were studied. The technique of whole plate fixation and staining with luxol fast blue and haematoxylin was employed to distinguish colonies and clusters into morphological groups. Retinoic acid at a concentration of 3 X 10(-7) M resulted in 97% inhibition of macrophage clonal growth and a mean increase in neutrophil clonal growth from bone marrow of 150 +/- 14%. These effects of retinoic acid were most obvious on bone marrow cluster forming cells. The inhibition of macrophage cluster growth was independent of exogenous colony stimulating factor. This dual action of retinoic acid is likely to be a direct effect on the progenitor cells since it is (1) independent of cell concentration, (2) not mediated by phagocytic cells or other bone marrow cells, and (3) was maximal in the first 2 d of incubation before the appearance of clusters.
Lorazepam was compared to placebo to assess its control of nausea and vomiting in patients receiving cytotoxic chemotherapy and prochlorperazine. The study design was a randomized, double-blind crossover in which three consecutive chemotherapy courses were compared so that each patient acted as his or her own control. Of 107 patients entered, 80 were evaluable for analysis. Lorazepam significantly reduced the severity and duration of nausea, the severity of vomiting, and the number of vomiting episodes when compared to placebo. Anxiety was reduced during lorazepam courses but not significantly when compared to placebo. There was significantly more sedation with lorazepam courses. Overall, patients preferred lorazepam courses although this preference was significant only in the patient subset receiving doxorubicin and cyclophosphamide. Lorazepam is a useful adjunct to prochlorperazine in patients receiving cytotoxic chemotherapy.
We have shown that in several mammalian cell lines a minor fraction of cytosine methylation is delayed for up to several hours after strand synthesis and that different methylases performed the immediate and the delayed classes of DNA methylation. To investigate the time course of this delayed methylation we have used three different cell lines, two of human and one of hamster origin. These were synchronized by two different methods: mitotic detachment and double hydroxyurea blocks. A uniform picture was obtained with all three cell lines. Delayed methylation of early replicating sequences occurred while cells were still in mid-S-phase, with the maximum rate of delayed methylation occurring in cells in the second half of S and in G2. Delayed methylation seems to be complete before cells entered the next G1-phase. Limited DNAase I hydrolysis of cell nuclei was used to test whether the delay in methylation in some DNA sequences was due to high levels of transcriptional activity. However, DNA sequences exhibiting delayed methylation showed no preferential concentration in or exclusion from DNAase I hypersensitive regions.
Explore the source record for details and available documents.
We have previously demonstrated that, while most enzymatic formation of 5-methylcytosine in the DNA of mammalian cells occurs very shortly after strand synthesis, there is also a minor fraction of methylation which occurs in some DNA sequences up to at least several hours after strand synthesis. Using a human cell line, we have examined the effects on these two classes of enzymatic DNA methylation of several compounds which have been reported to be inhibitors of methylation reactions. We have found that cycloleucine, ethionine, and 5'-deoxy-5'-methylthioadenosine (MTA) are all effective as inhibitors of enzymatic DNA methylation, but that there is no differential effect between the delayed and non-delayed methylation reactions. Tubericidin (7-deaza-adenosine) plus homocysteine inhibited delayed DNA methylation much more than non-delayed methylation (by up to 4 times). By contrast, 5-azacytidine produced a higher level of inhibition of DNA methylation at sites in the DNA in which the methylation occurred very shortly after strand synthesis. Also 5-azacytidine was by far the most potent inhibitor of DNA methylation of the compounds tested. S-Adenosyl-homocysteine and caffeine were found to have no effect on DNA methylation. These results are discussed in relation to the number and specificity of DNA methylases in these cells and to the cellular functions of those DNA sequences in which methylation is delayed for some hours after strand synthesis.