Characterization of Hodgkin's disease derived cell line HDLM-2.

The cell line HDLM-2 was established from the pleural effusion of a patient with Hodgkin's disease. Here, we describe the morphological, cytochemical, enzymological, immunological, molecular biological, and functional characteristics of the cell line. The results of this multiparameter profile show that HDLM-2 is different from other well-studied leukemia-lymphoma cell lines including other Hodgkin's disease derived cell lines. HDLM-2 cultures contain mainly mono- or binucleated cells, but also prominent giant cells with two to ten nuclei. HDLM-2 cells do not express an immunophenotype characteristic of a given cell lineage. However, the cells are positive for Ki-1, HeFi-1, Leu-M1, Tac, and HLA class II markers. Cytochemical, enzymological, and functional data are equally inconclusive, but are definitely not compatible with a monocyte/macrophage profile. Analysis of the gene status documents that T-cell receptor beta- and gamma-chain genes are rearranged while immunoglobulin heavy chain genes are in germline configuration. The combined results indicate a T-cell origin of HDLM-2 cells. The evidence available from this and other established Hodgkin's disease derived cell lines suggests a lymphoid origin of Hodgkin and Reed-Sternberg cells.

Reed-Sternberg cells in Hodgkin's cell lines HDLM, L-428, and KM-H2 are not actively replicating: lack of bromodeoxyuridine uptake by multinuclear cells in culture.

We compared the proliferation of mononuclear and multinuclear cells in four Hodgkin's cell lines, HDLM-1, HDLM-1d, L-428, and KM-H2, by examining their capacity to incorporate bromodeoxyuridine (BrdUrd) into nuclei. Approximately 5% of all cells in HDLM-1 cultures had two or more nuclei, a characteristic of Reed-Sternberg (RS) cells. Unlike mononuclear Hodgkin's (H) cells, these RS cells exhibited no uptake, or only minimal uptake of BrdUrd, suggesting that they did not replicate actively. Cytogenetic study showed that 25% of the HDLM-1 cells contained a tetraploid (4X) set of chromosomes with a characteristic two-peak distribution. Following treatment of HDLM-1 cells with phorbol ester, the percentages of 4X cells and RS cells increased to 50% and 12%, respectively. This increase in RS cells was not likely to be due to cell fusion as shown by the absence of hybridization of BrdUrd-positive and -negative nuclei. Phorbol ester has a short-term effect of blocking the exit of cells from G1 into S phase, but no effect on the transition from S phase to G2/M phase. The block is more prominent in 2X cells than in 4X cells, which may explain the increase in percentage of 4X cells in phorbol ester-treated cultures. In addition, phorbol ester induced the differentiation of H-RS cells, which was accompanied by loss of the marker HeFi-1 from the cell surface. Approximately one third of the RS cells did not express HeFi-1, or expressed only minimal amounts. The findings led us to the following conclusions: (1) The 4X cells probably are formed from 2X H cells as a result of disturbed cytokinesis, but not a cell fusion. (2) A considerable number of 4X cells were H cells, because the number of 4X cells consistently exceeded that of RS cells. (3) Since mitotic figures are extremely rare in RS cells and these cells did not show active BrdUrd uptake, the increased number of RS cells must also be a consequence of disturbed cytokinesis of H cells or a result of nuclear transformation (twisting, convolution, or separation of the nucleus) in H cells. (4) Most RS cells lose their proliferating capacity and some RS cells may undergo further differentiation. Uptake of BrdUrd and phorbol ester induction were also studied on the other three H-RS cell lines, HDLM-1d, L-428, and KM-H2, with results similar to those for HDLM-1.

Microcomputer-based image analysis systems for two-dimensional electrophoresis gels.

The intent of this overview is to provide the readers, especially those who are currently conducting two-dimentional electrophoresis, a basic understanding in the construction and use of microcomputer-based systems for the analysis of protein profiles generated by two-dimensional gel electrophoresis. In addition, a microcomputer-based system, employing fixed-point operations and effective algorithms, has been evaluated. The validity of this system has been demonstrated by using the two-dimensional silver-stained gels and fluorograms derived from the rat prostate. It is concluded that the present system can be used to aid the analysis of two-dimensional electrophoresis gels. An overall consideration of hardware and software components of a computer-based system is briefly discussed.

Extracellular matrix does not induce the proliferation, but promotes the differentiation, of Hodgkin's cell line HDLM-1.

Tumor cells from the Hodgkin's cell line HDLM-1 were cultured in extracellular matrix (ECM)-coated flasks. Within 24 hours, the cells underwent rapid differentiation, accompanied by morphologic and phenotypic changes. A slow and less prominent, but similar change was observed in HDLM-1 cells after they had been induced with phorbol ester for 5 days. The ECM-induced cells became HeFi-1-negative and expressed antigens 2H9 and 1E9 on their nuclear membranes. These cells had punctate acid-phosphatase and esterase activities. Approximately 70% of the ECM-induced cells were elongated and had long, blunt cytoplasmic projections. These findings, together with evidence the authors previously presented, indicate that H-RS cells are related to interdigitating reticulum cells. The authors believe that the ECM-induced HDLM-1 cells can be used as an important model for studies of the nature and cell lineage of Hodgkin's disease.

Hodgkin's disease derived cell lines HDLM-2 and L-428: comparison of morphology, immunological and isoenzyme profiles.

The cell lines HDLM-2 and L-428 were established from the pleural effusions of two patients with Hodgkin's disease. We studied and compared phenotypic characteristics of HDLM-2 and L-428 cells before and during induction of differentiation with 12-O-tetradecanoylphorbol 13-acetate (TPA) using a number of parameters. TPA-treated HDLM-2 and L-428 cultures did not show adhesion to plastic surface or aggregation of cells; the cells did not develop pseudopodia and were not phagocytic. Only a slight increase in the percentage of NBT-positive cells was observed for L-428 cells. TPA led to a cessation of cell proliferation and to a dose-dependent decrease in the number of viable cells in both cell lines. In HDLM-2 and L-428, treatment with TPA induced distinct morphological changes indicative of a partial differentiation along the myeloid cell lineage. In addition, the production and expellation of benzidine-positive, unnucleated particles were observed in HDLM-2 and L-428 cells. The induced isoenzyme profiles of carboxylic esterase and acid phosphatase resembled those found in myelomonocytic leukemia cell lines. Both cell lines were negative for immunological markers of the T- and B-cell lineages, but reacted with several markers associated with the myelomonocytic cell lineages. HDLM-2 cells produced a factor which could induce differentiation in 12 leukemia cell lines. The overall results suggest that Hodgkin and Sternberg-Reed cells constitute a unique cell type and might be derived from cells of the myelomonocytic cell lineage.

Marker profiles of human leukemia and lymphoma cell lines.

By means of the multiple marker analysis, a total of 55 human leukemia-lymphoma cell lines which included 15 T-cell, 30 B-cell, four myelomonocytic-cell, and six non-T, non-B cell lines was characterized for their marker profiles. The multiple markers used included a number of cell surface markers as detected by either rosette or immunofluorescence tests, enzyme assays, cytogenetic analysis, and certain functional assay. Based on the criteria previously defined it was found that all the cell lines were proved to represent original leukemia-lymphoma of ALL, AML, CLL, CML in blastic phase or variety of lymphomas. The monoclonality, a "frozen" state at a specific state of differentiation-maturation, and cytogenetic marker in each leukemia-lymphoma cell line were remarkable common properties and were stable for years of cultivation. Similar, if not identical, general characteristics were observed in the study on 344 cases of uncultured fresh leukemia-lymphomas by the multiple marker analysis. While no single marker specific to any type of tumor was found, the study offers not only a basis for better understanding of the biology of leukemia-lymphoma but also an insight into normal hematopoietic cell differentiation in man.

Malignant lymphoma of pre-T-cell type terminating in acute myelocytic leukemia. A case report with enzymic and immunologic marker studies.

We report a case of a T-zone malignant lymphoma of a cervical lymph node developing in a 25-year-old man. Only 14% of the marrow was originally involved, but within two months massive, leukemic dissemination ensued. The blast cells were unable to bind sheep erythrocytes (E) but expressed human thymus leukemia antigen (HTLA) and common ALL-stem-cell (cALL) antigen and had high terminal deoxynucleotidyl transferase (TdT) and acid phosphatase activity. These findings suggest a malignant lymphoproliferative disorder of pre-T-cell type. Complete remission was achieved with intensive chemotherapy. Two months later, acute myelomonocytic leukemia was diagnosed; at this time, over 90% of the blast cells were peroxidase, sudan black, and chloracetate-esterase positive. Consistent with loss of high TdT activity and HTLA and cALL antigens, 86% of the blasts now expressed Ia-like antigens. Cytogenetic studies demonstrated hyperdiploidy. Reports of granulocytic leukemia in lymphoma are reviewed in the context of the above findings and the hypothesis that a leukemogenic factor affects a multipotential stem cell.

Establishment and characterization of human B-lymphocytic lymphoma cell lines (BALM-3, -4 and -5); intraclonal variation in the B-cell differentiation stage.

This study describes the establishment of three non-Burkitt B-lymphoma cell lines (BALM-3, BALM-4 and BALM-5) originating from the pleural effusion of a patient with a poorly differentiated diffuse lymphocytic lymphoma. The cells of BALM-3, -4 and -5 exhibited a number of properties which distinguish them from the usual B-cell type lymphoblastoid cell lines. Thus, they lacked the Epstein-Barr virus genome and had abnormal chromosome constitutions including a 14q+ marker. The presence of the identical surface immunoglobulin isotypes (gamma and chi chain determinants), and Ia-like B-cell-associated antigen in the cultured cells and in the "fresh" lymphoma cells in vivo was demonstrated. These findings strongly suggested that these cell lines have B-cell characteristics and were derived from the original tumor cell population. BALM-5 cells, however, showed somewhat different growth, cell surface marker profile and functional characteristics compared to those of BALM-3, and -4 cells. These variations suggest that the BALM-5 cells were probably at different stages of B-cell maturation than those of BALM-3 and -4, even though all three cell lines (established in three separate flasks) originated from the cells of the same pleural effusion of a lymphoma with monoclonal B-cell characteristics.