Chromatographic forms of terminal deoxynucleotidyl transferase in normal lymphoid cells and in leukemia cells at presentation and relapse

Normal thymocyte and bone marrow terminal deoxynucleotidyl transferase (TdT) have distinguishing characteristics by phosphocellulose chromatography in Tris buffer: marrow TdT elutes as a single peak at 0.3 M salt, whereas thymocyte TdT separates into two forms, one at 0.3 M salt and one at 0.4 M salt. Since the majority of TdT-positive acute leukemias are anatomically bone marrow-derived, one would have predicted the presence of a bone marrow TdT-phosphocellulose chromatographic pattern in such patients. However, in 376 consecutive, untreated TdT-positive acute lymphoblastic leukemias (ALL) studied by us we have invariably encountered the two-peak thymocyte-type phosphocellulose pattern. The TdT patterns in the thymic-dependent, TdT-positive lymphoma of AKR mice, and the TdT-positive bone marrow-derived, thymic-independent Abelson virus leukemia of Balb/C mice duplicate the situation in human ALL: a thymocyte pattern is seen in both the marrow-derived and thymus-derived diseases. This chromatographic difference between leukemia-associated and normal marrow-associated TdT in both murine and human leukemia suggested that phosphocellulose-TdT patterns might be useful for monitoring residual marrow tumour cell burden in TdT-positive leukemia. This has not turned out to be the case: in eight patients studied in early relapse the blast cell TdT pattern was the single-peak 0.3 M species. Therefore, leukemic cell TdT cannot reliably be distinguished from normal marrow cell TdT. The chromatographic behaviour of TdT may be regulated by phosphorylation-dephosphorylation, the 0.3 M salt peak can be converted to the 0.4 M salt species by treatment with protein kinase and ATP, and the 0.4 M species can be converted to the 0.3 M form by exposure to alkaline phosphatase. Thus, apparently anatomic compartment-specific forms of TdT may only reflect differing cellular metabolic activity.     

Glucocorticoid receptors in leukemia cells: An appraisal

There is growing interest in the molecular events which govern response to therapy in the leukemias. Glucocorticoid therapy would appear to be a fertile area for study as much is known about mechanism of steroid hormone action. In this paper we review current knowledge of glucocorticoid receptor numbers and function in leukemic blast cells and their relationship to steroid treatment and outcome.     

胸腺粘膜相关淋巴瘤（病例报告及文献复习）

本文报道经免疫组化证实的8例B细胞性胸腺粘膜相关淋巴瘤,其特征为胸腺组织结构被中心细胞样小淋巴细胞浸润,但Hassall's小体仍可部分保存,并常形成含有分泌物的微囊,组成典型的淋巴上皮样病变,符合粘膜相关淋巴组织淋巴瘤的特征。免疫组化反应显示多数为MB2单抗反应阳性、λ或κ阳性的B细胞,属低度恶性淋巴瘤,对该瘤的组织发生,细胞成份,诊断和鉴别诊断进行了讨论,并指出应从胸腺瘤淋巴细胞型中单独分出来。     

Properties of an anti-idiotype antiserum: Demonstration of cross-reacting mitogenic determinants on bone marrow plasmacytoma cells

An antiserum against a monoclonal protein of 1gG/κ type from a patient S. was produced by immunization of guinea pigs. Idiospecificity of the antiserum (anti-Id AS) was tested by immunoprecipitation and hemagglutination inhibition. About 70% mononuclear cells prepared from the bone marrow (BM) aspirate of this patient stained positive when the antiserum was used in an indirect immunofluorescence (IF) procedure detecting surface membrane structures. Commercial antisera specific for γ, κ and λ immunoglobulin (Ig) chains stained only 5–17% of the cells. With μ chain and δ chain specific antisera no reactivity could be observed. The presence of idiotype cross-reacting determinants (Id-CR-D), that is idiotypes not associated with Ig isotypes could be also demonstrated by a significant increase of ³H-thymidine incorporation of the BM cells in the presence of the anti-Id AS. Phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A had no effect. In a cell sorting experiment using affinity gel chromatography it could be shown that the anti-Id AS preferentially binds to proplasmacytes. Cells of this type were enriched two-fold over plasma cells in the eluate. The analysis of peripheral blood lymphocytes (PBL) from the patient revealed about 40% positive cells without correlation to a certain subpopulation. Administration of prednisolone to the patient resulted in a significant reduction of the Id positive cells within two days. In conclusion it appears that our antiserum detects cells of an individual tumour clone within PBL and BM-cells. By the stimulating effect of the anti-Id AS a proliferative potential of plasmacytoma cells could be defined which may work also ‘in vivo’. Thus, Id-CR-D may operate as a tumour growth receptor.     

Changes in the expression of class I major histocompatibility complex antigen RNA induced by interferon in rat hepatoma cells

The rat hepatoma cell line 17X was studied to determine if it expressed major histocompatibility complex (MHC) class I antigen RNA and to see if interferon treatment would affect its expression. Under normal cell culture conditions, MHC class I RNA in 17X hepatoma cells is virtually undetectable. Administration of rat interferon at a concentration of 1000 units/ml for 48 h to 17X cells in culture resulted in the appearance of detectable levels of RNA for MHC class I antigens. The interferon-induced increase in class I RNA was accompanied by de novo synthesis of immunoprecipitable, metabolically radiolabeled class I antigens in the 17X hepatoma cells.     

Bone marrow and extramedullary variations of cell membrane antigen expression in childhood lymphoid neoplasias at relapse

To increase our knowledge of the pathogenesis of tumour recurrence, cell membrane phenotypes were determined on bone marrow and extramedullary tumour cells at diagnosis and at relapse in 24 children with lymphoid malignancies. There were 19 patients with acute lymphoblastic leukemia and five with non-Hodgkin's lymphoma. Membrane characteristics used for classification were E-rosetting, T antigen, surface immunoglobulin (sIg) and Ia antigen. Twelve patients were phenotyped as non-T, non-B, five as T-like and seven as B-like leukemia/lymphoma. Eighty-eight tissue samples were assayed for cell surface markers at the time of relapse(s). Lymphoblasts from 18 children (75%) demonstrated no variation in membrane marker expression. Six patients (25%) showed alteration of antigen expression on lymphoblasts obtained during relapses. This was manifested by loss of Ia antigen in two patients, loss of E-rosetting in one patient and loss of sIgD in one patient. Lymphoblasts from two patients, initially non-reactive with the four membrane markers utilized, expressed Ia antigen on subsequent relapses. Despite these variations no patient was categorized differently (i.e. T-like becoming non-T, non-B). Simultaneous lymphoblast phenotype determination from multiple body sites in 13 children showed no variation in marker analysis. A lymphoblast's phenotype remains stable throughout repeated relapses and is not influenced by relapse site in most children with lymphoid neoplasias. This information may be helpful in designing protocols where cytotoxic monoclonal antibodies are used as a treatment modality in patients with recurrent disease.     

A cyclic AMP binding protein pattern useful as a biochemical differentiation marker of erythroleukemic cell lines and normal cloned erythroblasts

Previous study of a variety of a human hemopoietic cell lines and normal samples with 8-N₃-³²p-cAMP photoaffinity labelling, SDS-polyacrylamide gel electrophoresis and autoradiography revealed five distinct cAMP binding protein patterns, each of which were restricted to cells of particular lineages. One pattern unique to K562 cells was characterized by the presence of three bands designated a, b and d. In order to ascertain the significance of this finding, we studied by the same methods normal human erythroblasts harvested from methylcellulose cultures of fetal liver and adult blood, and murine and human erythroleukemic cells (MEL and HEL) as well as K562, cultured with and without chemical inducers of erythrodifferentiation. Three band patterns recognizable as similar to that of K562, but distinctive from those of myeloid and lymphoid cells, were noted of cells of erythroblastic origin. Both HEL and MEL, as well as the normal erythroblasts, exhibited heaviest labelling of band a in contrast to K562, which exhibited heaviest labelling of band d. Relative labelling of band b and d of HEL and MEL increased following treatment with 50 μ hemin or 4 mM hexamethylenebisacetamide (HMBA), respectively. Treatment of K562 with hemin, however, resulted in increased band a. Thus, among hemopoietic cells, an isozymic cAMP binding protein pattern has been identified which is characteristic of the erythroid lineage. The relative abundances of the three components (a, b and d) have been furthermore noted to undergo a series of shifts during induced differentiation. Cyclic AMP binding proteins may thus prove useful as a biochemical marker system in the recognition and analysis of erythroid differentiation.     

Reversal of doxorubicin-resistance in sarcoma 180 tumor cells by inhibition of different resistance mechanisms

Resistance of tumor cells to doxorubicin is a multifactorial phenomenon. In the present investigation, the ability of resistance modifiers against different resistance mechanisms was analysed. Substances which block P-glycoprotein (P-170) function circumvented resistance of doxorubicinresistant sarcoma 180 (S180) cells completely (verapamil, thioridazine) or partially (hycanthone), whereas inhibitors of glutathione S-transferase (ethacrynic acid, N-ethylmaleimide, buthionine sulfoximine), and protein kinase C (staurosporine, acridine orange) caused only a partial reversion of resistance. In contrast, an inhibitor of alkaline phosphatase (levamisole) did not overcome doxorubicin-resistance. These results indicate that P-glycoprotein blockers might be more effective to modulate doxorubicin-resistance of S180 cells as compared to other modifiers. Further investigations using other MDR cell lines are required to clarify the generality of these findings.     

Skeletal changes in childhood acute lymphoblastic leukemia: Correlation with the presence of the cALL antigen

We studied the occurrence of the radiolucent metaphyseal bands in pretreatment skeletal X-rays and the phenotype of leukemic cell in 66 children with non-T, non-B ALL. A striking correlation was found between the expression of the CALLA on the leukemic cell and the occurrence of the early metaphyseal bands. We speculate that the bands might reflect an ongoing anti-leukemic reaction which may have been in progress before the disease became clinically manifest and which is associated with relatively favorable prognosis after even a modest chemotherapy.     

Clinical significance of quantitative analysis of carcinoembryonic antigen assessed by flow cytometry in fresh human gastric cancer cells

The expression of carcinoembryonic antigen(CEA) on tumor cells freshly excised from 51 patients with gastric cancer was studied using flow cytometry. The expression of CEA by flow cytometry was more quantitative than that by immunohistochemical staining. There was no relationship between the fluorescence intensity assessed by flow cytometry and serum CEA levels, except for patients with a high titer of serum CEA. The patients with high grade CEA expression on tumor cells by flow cytometry had poor prognoses, compared to patients with low CEA expression in undifferentiated gastric cancer. Thus, it is suggested that the quantitative CEA expression on tumor cells by flow cytometry could be a useful prognostic marker in postoperative gastric cancer patients.     

Candidate counterparts of sezary cells and adult T-cell lymphoma-leukaemia cells in normal peripheral blood: An ultrastructural study with the immunogold method and monoclonal antibodies

The ultrastructural and immunologic features of normal convoluted T-lymphocytes were studied with a panel of monoclonal antibodies and the immunogold technique and these were compared with cells from patients with Sézary syndrome (SS) and adult T-cell lymphoma-leukaemia (ATLL). According to the characteristics of the nucleus, two distinct T-cell subtypes, representing 2–4% of normal peripheral blood lymphocytes were recognized: (i) a cerebriform lymphocyte (‘Sézary-like’) characterized by narrow and deep nuclear indentations, closely resembling the cells of SS, and (ii) a convoluted or polylobated lymphocyte (‘ATLL-like’), with shorter and broader nuclear indentations than those seen in SS, that resemble the cells of ATLL. Both types of lymphocytes were positive with the monoclonal antibodies OKT3, OKT4, OKT17 and FMC3 and were negative with OKT8, OKM1 and FMC4 (anti-HLA-Dr) as SS and ATLL cells. A difference was observed with the expression of the anti-T monoclonal antibody 3A1: Sézary-like lymphocytes, like SS and ATLL cells, were 3A1 negative whilst ATLL-like lymphocytes were 3A1 positive. The close morphological and membrane phenotype similarities observed between these two types of lymphocytes and the cells from SS and ATLL suggest that they may well represent the normal counterparts of the malignant T cells in both conditions.     

Differential methylation of class 1 histocompatibility antigen genes in T-cell lines derived from two different types of T-cell malignancies

We have previously shown that two human T-cell lines (HSB and 8402) derived from patients with childhood T-cell ALL (T-ALL) do not synthesize detectable mRNA for HLA-DR. The DR genes in both cell lines are hypermethylated relative to the same genes in T-cell lines infected with human T-cell leukemia virus (HTLV) and derived from patients with adult T-cell leukemia/lymphoma (ATL). These latter cell lines do express HLA-DR-mRNA, as well as HLA-DR surface antigens. We report here that the genes for HLA class I antigens are also highly methylated in the T-ALL T-cell lines relative to the same genes in the ATL T-cell lines, normal peripheral blood T cells, and autologous normal B-cell lines. In spite of substantial differences in the extent of methylation of class I-related genes, no obvious differences exist among these cell types in their levels of expression of HLA-A and -B antigens. The data clearly indicate, however, that the class I and class II components of the major histocompatability complex are unusually hypermethylated in several T-ALL-derived cell lines, while ATL T-cell lines do not substantially differ in this respect from normal peripheral blood T cells.     

Evidence for the induction of protease activity on cultured tumour cells as a consequence of implantation into nude mice

We have studied the enzymic status of a tumour associated protease on human colonic tumour cells cultured in vitro and grown in vivo in nude mice. The cultured tumour cells lack this protease. The cells of the tumour colonies in the mice possess active enzyme although the tissue contains an inhibitor capable of inactivating this tumour cell protease. The evidence indicates that the induction of this protease on the tumour cells in a consequence of implantation of the cultured cells into the nude mouse.     

The evaluation of low dose hyper-radiosensitivity in normal human skin

BACKGROUND AND PURPOSE: The laboratory phenomenon of low dose hyper-radiosensitivity (LDHRS) describes an excess of cell kill at doses below 1Gy relative to that predicted by the linear quadratic model. These data have stimulated clinical investigation into LDHRS in vivo. PATIENTS AND METHODS: Skin was used as a model of normal human tissue. Two studies were initiated investigating the response to low doses of radiation. Study 1 compared once daily skin doses of approximately 0.5 and >1.0Gy in 24 patients receiving pelvic radiotherapy. Skin biopsies before and during radiotherapy were analysed histologically to assess the basal cell density (BCD). Study 2 compared two regimens of equal dose/time intensity--an ultrafractionated regimen (0.5Gy TDS x 12 days) with a conventional regimen (1.5Gy OD x 12 days). Skin biopsies taken during treatment assessed BCD and proliferative index. In both studies the changes in BCD were compared using non-linear regression analysis. RESULTS: Study 1. The results show a significantly greater reduction in BCD in the low dose group when BCD is plotted against dose. This effect is lost when BCD is plotted against time Study 2. The results demonstrate a significantly greater reduction in BCD in the higher dose/fraction arm. The proliferative response was similar in both treatment groups. CONCLUSIONS: These data suggest that LDHRS does not occur in skin following doses of approximately 0.5Gy/fraction when regimens of equal dose/time intensity are compared. As only small volumes of normal tissue were irradiated it is difficult to predict the biological relevance of this with respect to larger field low dose per fraction irradiation regimens or risk of cancer induction. Equally we cannot extrapolate to effects resulting from exposure to doses <0.5Gy or to the effects of low doses on other endpoints.     

Induction of hemoglobin synthesis in dimethylsulfoxide-treated friend erythroleukemic cells grown in the presence of cytochalasin B

The experiments reported here were designed to determine the necessity for nuclear and/or cell division for erythroid differentiation in the presence of the inducer, dimethylsulfoxide (Me₂SO). Murine erythroleukemic cells are induced to synthesize hemoglobin only when the cells are cultured at a density which allows them to replicate in the presence of Me₂SO. Hemoglobin is not detected in the cytoplasm of the cells prior to 48 h. Cells grown in the presence of inducer (Me₂SO)+cytochalasin B (CB) do not divide, but do undergo karyokinesis thereby increasing in size as they become multinucleated. Under these experimental conditions, the CB-treated cells are also capable of synthesizing hemoglobin. Measurements of the rate of hemoglobin synthesis suggest that induced CB-treated cells accumulate more hemoglobin per induced cell than do cells induced by Me₂SO alone. These results indicate that: (a) while cytokinesis is not required to induce hemoglobin synthesis, the ability of the cells to replicate their genomes is critical, and possibly allows Me₂SO to interact with the genome to initiate the erythroid cell program; (b) CB does not alter the time course of hemoglobin synthesis; and (c) the increased levels of hemoglobin in the CB-treated cells is probably related to the increased cell-size mediated by the mold metabolite, and not to the induction of a higher percentage of cells.     

Kinetics of colony-forming cells (CFU-c) in adult acute leukemia and their prognostic relevance

The in vitro growth pattern of bone marrow and peripheral blood in soft-agar cultures was studied in 50 previously untreated patients with adult acute leukemia. Patients were followed from time of first diagnosis, during induction treatment, in remission at various time intervals, at relapse and during subsequent re-induction therapy. The distribution of granulopoietic progenitor cells was analysed to determine their prognostic significance for remission incidence, remission duration and survival. All patients revealed an abnormal growth. ANLL patients showing a decreased clone number in the marrow but an increased number of clones in the peripheral blood, had a significant higher probability to enter remission and a significant longer remission than those having clones within normal range at first presentation. On the contrary, ALL patients responding to induction treatment, had a better colony and cluster growth in the bone marrow than those failing to respond. No significant correlation was found between in vitro growth and survival. It is concluded that colony-forming cells of both bone marrow and peripheral blood seems to be of some value in predicting the response rate and length of remission in ANLL and ALL, and in possibly selecting patients with a high chance to respond to current cytostatic regimens.     

Modulation of the reversibility of actinomycin D cytotoxicity in HeLa cells by verapamil

Actinomycin D treatment (0.001–0.005 μg/ml; 0.5–24 h) induced a dose and time response shifting of nucleolar to nuclear fluorescence. In the presence of verapamil, cells were more responsive to actinomycin D. Translocation of protein B23 occurred with lower doses of actinomycin D and in shorter incubation times in the presence of verapamil. Short exposure (0.5 h) of HeLa cells to actinomycin D (0.05–0.25 μg/ml) induced ‘reversible’ translocation of protein B23 as well as ‘reversible’ inhibition of cell growth and RNA synthesis. Verapamil (5 μM) included in the cell culture after removal of actinomycin D inhibited the recoveries of cell growth, RNA synthesis as well as the corresponding relocalization of protein B23 from the nucleoplasm to nucleoli. These results indicate that verapamil can potentiate the antiproliferating activity of actinomycin D by inhibiting reversibility of its cytotoxicity and suggest clinical application.     

Dissociation of interleukin-2-mediated cell proliferation and interleukin-2 receptor upregulation in adult T-cell leukemia cells

We studied cell proliferation and interleukin-2 (IL-2) receptor upregulation mediated by exogenous IL-2 in leukemic cells from adult T-cell leukemia (ATL) patients to characterize some aspects of abnormal IL-2 receptor expression in ATL. Leukemic cells from 7 ATL patients examined showed no or very poor proliferative response to IL-2 though they expressed IL-2 receptors without any stimulation. In contrast, ATL leukemic cells cultured with IL-2 for 2 days expressed about twice as many IL-2 receptors as those of cells cultured without IL-2. Thus, in ATL leukemic cells, there seems to be a dissociation between the signal(s) for two different effects mediated by IL-2, cell proliferation and IL-2 receptor upregulation.     

Ribonucleotide reductase activity and growth of glutathione-depleted mouse leukemia L1210 cells in vitro

L1210 cells treated with -buthionine-(S/R)-sulfoximine (BSO) had glutathione (GSH) and non-protein thiol levels only 15% that of control. These GSH-depleted cells grew as well as the control L1210 cells and there was no decrease in ribonucleotide reductase activity in situ as measured by the conversion of [¹⁴C]cytidine to deoxytidine nucleotides and incorporation into DNA. Further, when these BSO-stressed cells were treated with hydroxyurea or IMPY, there was no potentiation of the inhibition caused by hydroxyurea or IMPY alone. These data indicate that the glutathione/glutaredoxin system of ribonucleotide reductase is not the sole carrier of reducing equivalents from NADPH for the reduction of the 2′-position of the corresponding ribonucleoside 5′-diphosphate; and that glutathione is not critical in regenerating the tyrosyl free-radical on the M2 subunit which is destroyed by the hydroxyurea or 2,3-dihydro-1H-pyrazolo-[2,3-a]imidazole (IMPY) treatment.