Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16.

In the endeavour to develop a model for studying gene therapy of cancers associated with human papillomaviruses (HPVs), mouse cells were transformed with the HPV type 16 (HPV16) and activated H-ras oncogenes. This was done by cotransfection of plasmid p16HHMo, carrying the HPV16 E6/E7 oncogenes, and plasmid pEJ6.6, carrying the gene coding for human H-ras oncoprotein activated by the G12V mutation, into secondary C57BL/6 mouse kidney cells. An oncogenic cell line, designated MK16/1/IIIABC, was derived. The epithelial origin of the cells was confirmed by their expression of cytokeratins. No MHC class I and class II molecules were detected on the surface of MK16/1/IIIABC cells. Spontaneous metastases were observed in lymphatic nodes and lungs after prolonged growth of MK16/1/IIIABC-induced subcutaneous tumours. Lethally irradiated MK16/1/IIIABC cells induced protection against challenge with 10(5) homologous cells, but not against a higher cell dose (5 x 10(5)). Plasmids p16HHMo and pEJ6.6 were also used for preventive immunization of mice. In comparison with a control group injected with pBR322, they exhibited moderate protection, in terms of prolonged survival, against MK16/1/IIIABC challenge (P < 0.03). These data suggest that MK16/1/IIIABC cells may serve as a model for studying immune reactions against HPV16-associated human tumours.

[Selective photodynamic destruction of leukemic cells]. / Selektivní fotodynamická destrukce leukemických bunek.

BACKGROUND: Residual leukemic cells if present in autologous bone marrow grafts or CD34+ concentrates obtained from peripheral blood may increase the risk of relapse after autotransplantation. We are presenting the employment of a new method which was introduced into the photodynamic therapy, namely enhancement of synthesis of the photosensitizing compound, protoporphyrin IX, in cancer cells, following application of its metabolic precursor, 5-aminolevulinic acid, for the specific destruction of leukemic cells. METHODS AND RESULTS: By determining cell viability using tetrazolium salt reduction (MTT), by flow cytometrypropidium iodide assay and by determining cell proliferation using bromodeoxyuridine incorporation we studied the effect of photodynamic therapy based on the application of 5-aminolevulinic acid on the cells of leukemic cell lines HL60 (human promyelocytic leukemia), HEL (erythroleukemia), DAUDI (B-cell leukemia), JURKAT (T-cell lymphoma), blast cells of patients with acute myelogenous leukemia as well as on normal lymphocytes and normal human bone marrow progenitors. In in vitro experiments photodynamic therapy based on an administration of 5-aminolevulinic acid (1 mM, 4 h, 18 J/cm2) lowered the number of viable leukemic cells by over 2 orders (with the exception of HEL cells) and eliminated blast cells in mononuclear cell preparations of six out of seven patients with acute myelogenous leukemia. On the other hand the viability of normal resting lymphocytes was little affected by photodynamic therapy (number of necrotic cells increased from 6 to 11%) and also the clonogenic activity of the progenitor cells of normal bone marrows did not decrease substantially (CFU-GM to 60% and BFU-E to 55% of the original activity). CONCLUSIONS: Photodynamic therapy based on the application of 5-aminolevulinic acid is a perspective method for the specific destruction of leukemic cells in autologous transplants.

Selective destruction of leukaemic cells by photo-activation of 5-aminolaevulinic acid-induced protoporphyrin-IX.

The effect of 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) on the viability and proliferation of leukaemia/lymphoma cells as well as normal human lymphocytes has been investigated by flow cytometry-propidium iodide assay (FC-PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) incorporation and on clonogenic activity of normal human bone marrow progenitor cells by clonogenic methods. ALA-PDT (1 mM 5-ALA, 4 h, 18 J cm-2) reduces the number and/or suppressed proliferation of leukaemic cells of promyelocytic (HL60), B-cell-derived (DAUDI) and T-cell-derived (JURKAT) cell lines by 2 logs and that of the HEL erythroleukaemia cells by 77%. The effect of ALA-PDT on quiescent human lymphocytes is small (85% viable cells after ALA-PDT). The proliferation of lymphocytes subjected to ALA-PDT and induced with phytohaemagglutinin (PHA) decreases by 75% as compared to the untreated control. For normal human bone marrow progenitors, 58% of colony-forming units-granulocytes-macrophages (CFU-GM) and 55% burst-forming units-erythrocytes (BFU-E) activities are preserved.

Transplantation of allogeneic peripheral blood progenitor cells--a single centre experience.

13 patients have been transplanted at Institute of Hematology and Blood Transfusion since 1995 using allogeneic PBPC either alone or with bone marrow as a source of progenitor cells. All donors were G-CSF mobilised HLA identical family members. PBPC harvests were performed on D 4,5, (6) of G-CSF administration. The medium content of TNC, CD34+, CD3+, CD4+and CD8+cells/kg b.w. of the recipients in the grafts were: 13,1x10(8)(TNC), 11,4x10(6)(CD34+), 393x10(6)(CD3+) 243x10(6)(CD4+), 125x10(6)(CD8+) The patients received either BuCy2 or CyTBI preparative regimen and Cyclosporin A + short course of Methotrexate for GVHD prophylaxis. Engraftment of ANC >500 was achieved by D+16 and PLT >20.000 by D+19. Three of ten evaluable patients developed acute and three of nine chronic GVHD. 8 patients survive with the longest follow up 776 days.

Photodynamic effects of meso-tetra (4-sulfonatophenyl)porphine on human leukemia cells HEL and HL60, human lymphocytes and bone marrow progenitor cells.

Meso-tetra(4-sulfonatophenyl)porphine (TPPS4), in combination with a light dose of 14 J cm-2, has a profound negative effect on the proliferation and viability of leukemia cells HL60 (human promyelocytic leukemia) and HEL (human erythroleukemia), the viability of normal lymphocytes and the colony-forming activity of human bone marrow progenitor cells. However, normal leukocytes (monocytes, granulocytes) are, to a large extent, resistant to photodynamic treatment (PDT). Whilst DNA fragmentation suggesting apoptosis is induced in HL60 cells, accumulation in the interphase of the cell cycle (G0/G1, G2/M) is mainly operative in the TPPS4-mediated PDT of HEL cells. The "dark" effect of TPPS4 on the cell viability is below 15% up to a concentration of 40 microM.

Immunophenotypic significance of the "lymphoid" CD38 antigen in myeloid blood malignancies.

In 72 patients with blood malignancies (leukemias), the expression and distribution of the "B-lineage" antigen CD38, was analyzed, individually and in combination with CD19, CD10, HLA-DR, CD13, CD14, CD33, CDw65, CD2 and CD7. The expression of CD38 on the surface of leukemic cells was totally different from its expression on normal hematopoietic cells. Its positivity in myeloid malignancies was as follows: In patients with acute myeloid leukemia in 21/28 cases-75% (probability of expression 0.68 +/- 0.2, p < 0.05) and in patients with chronic myeloid leukemia in 4/6 cases-66%. In lymphoproliferative malignancies the CD38 antigen was expressed as follows: In patients with acute non-T lymphoblastic leukemia in 12/16 cases-75% (probability of expression 0.7 +/- 0.2, p < 0.05) and in patients with chronic B lymphocytic leukemia in 6/8 cases-75%. CD38 was also found positive in patients with acute mixed lineage leukemia.

Expression of CDw12 and CD17 cell surface antigens on leukemic cells from patients with blood malignancies.

In 67 cases of newly diagnosed blood malignancies, NonT-ALL, T-ALL, AMLL, AML, CML, CLL, HCL, PLL, MDS, B splenic lymphoma, AUL, as well as in 9 cell lines (U937, HEL, Jurkat, HL60, UHKT2, KG1, Raji, K562, REH), we have analysed the expression and distribution of 2 relatively incompletely studied antigenic markers from the CD nomenclature: CDw12 and CD17, individually and in combination with well characterized ones. We present our data for the usefulness of these molecules in immunodiagnosis of leukemias and lymphomas.