Changes in antigen expression on human FME melanoma cells after exposure to photoactivated hematoporphyrin derivative.

Exponentially growing melanoma cells of the line FME were incubated with hematoporphyrin derivative (HpD) for 1 and 18 h and subsequently exposed to light in the presence of HpD. Quantitative changes in the expression of the melanoma-associated surface antigen p250 recognized by the monoclonal antibody 9.2.27 were studied by flow cytometry. Treatment with HpD and light resulted in no immediate changes in the antigen expression. However, a few hours after light exposure a significant reduction in antigen expression was observed. For cells incubated with HpD for 1 h, the minimum expression of the antigen was observed 6 h after the irradiation, and the duration of the reduced expression was almost dose independent. On the other hand, the duration of the reduced antigen expression increased strongly with light dose for cells incubated with HpD for 18 h. In both cases antigen expression decreased exponentially with the product of drug concentration and light dose, indicating that there is no rapid mechanism by which the cells can repair the damage which leads to reduced antigen expression. Days were needed before the cells expressed a normal level of the antigen. A slight overshoot of the level of antigen expression above that for untreated cells was observed 2-5 days after light exposure depending on the incubation conditions with HpD and the light dose. At a given cell survival level (greater than 0.1), the decrease in antigen expression was more pronounced on cells incubated with HpD for 1 h than on cells incubated with the drug for 18 h.

Is the RPM cell line a useful model for the study of megakaryocyte development?

The differentiation of megakaryoblasts into megakaryocytes and their release of blood platelets are complex and poorly understood processes. As an aid to investigate this process several cell lines with megakaryocyte characteristics have been established. One of these cell lines is the rat promegakaryoblast-like (RPM) cell line established by Cicoria and Hempling and used by others to describe maturation processes in megakaryocytes. We have used this cell line to study the synthesis of platelet-specific marker proteins. Severe difficulties led us to perform control experiments to confirm earlier findings. We were unable to confirm several of the previous reports, and we conclude that this particular cell line should not be recommended for the study of megakaryoblast differentiation.

Comparison of extracellular matrix in human osteosarcomas and melanomas growing as xenografts, multicellular spheroids, and monolayer cultures.

BACKGROUND: The composition of extracellular matrix in human xenografts and spheroids were compared with the monolayer cultures from which they originated. Collagen I, fibronectin, acetylglucosamine, and acetylgalactosamine were quantitated in two osteosarcomas and one melanoma. METHODS: Using fluorescence microscopy, extracellular matrix constituents in the cellular and extracellular compartment were measured, whereas flow cytometry measured the extracellular matrix constituents bound to the cell surface as well as the total cellular amount including intracellular and surface bound constituents. RESULTS: The fluorescence microscopy measurements, demonstrated that the xenografts contained more or equal quantities of the extracellular matrix constituents compared with the spheroids. Flow cytometric measurements of total cellular amounts, showed that cells from xenografts usually contained more or equal amounts as the spheroid cells, which contained less or equal amounts as the monolayer cells. The surface expression of the extracellular matrix constituents increased or there were no significant differences, comparing cells grown as monolayers, spheroids, and xenografts. CONCLUSIONS: The data shows that multicellular spheroids being an in vitro system of intermediate complexity between monolayer cultures and tumours, contain an extracellular matrix corresponding to some degree to this intermediate position.

Flow cytometric measurements of heat shock proteins.

A fixation and immunofluorescence staining procedure for measurements of heat shock proteins (hsp) by flow cytometry is reported. Three fixatives were compared: 80% methanol at -20 degrees C for 1 h, 70% ethanol at 0 degree C for 1 h, and 3% paraformaldehyde at 4 degrees C for 1 h followed by 0.2% NP-40. Cells fixed with methanol showed strongest immunofluorescence and lowest nonspecific fluorescence. The level of hsp 70 as a function of time after heating followed the same kinetics as the development of thermotolerance reported by others. The level of hsp 70 increased with increasing heat dose up to a maximum heat dose, and above this heat dose a decrease in the level of hsp 70 was observed. Correlated measurements of the level of hsp 70 and DNA showed that hsp 70 was found in all phases of the cell cycle. The level of hsp 70 increased about two-fold in unheated cells throughout the cell cycle. The increase in G2 + M cells compared with G1 cells was lower in cells heated at 45 degrees C for 20 min followed by incubation at 37 degrees C for 24 h before fixation and staining, than in unheated cells. The results show that flow cytometry provides a rapid and quantitative technique for measuring hsp. Correlated measurements of hsp and other cellular parameters might also be obtained.

Expression of melanoma-associated antigen of thermotolerant human cells.

The expression of the melanoma-associated antigen p250 on thermotolerant cells and the effect of a second heat dose on the antigen expression have been measured by flow cytometry. The human melanoma cell line FME was heated at 43.5 degrees C for 120 min after a priming heat dose at 43.5 degrees C for 20, 40 or 60 min. Cells preheated at 43.5 degrees C for 40 and 60 min followed the same kinetics of development and decay of thermotolerance, with maximum thermotolerance 16 h after the priming heating, and the thermotolerance had decayed by 48 h. Cells preheated at 43.5 degrees C for 20 min showed maximum thermotolerance after 7 h and decay by 24 h. Heat reduced the expression of the melanoma-associated antigen in a dose-dependent manner. Thermotolerant cells were given a second heat dose (43.5 degrees C for 120 min) and the antigen expression measured immediately after heating. Fractionated hyperthermia using the lower predose (43.5 degrees C for 20 min) might have an additive effect on the reduction of antigen expression, while the highest predose (43.5 degrees C for 60 min) protected against reduction in antigen expression. The development and decay of resistance against heat-induced reduction in expression of melanoma-associated antigen followed a similar time course as thermotolerance in terms of cell survival. Maximum resistance was observed 12 h after the priming heat treatment, and the resistance had decayed by 48 h.

Hyperthermia-induced shedding and masking of melanoma-associated antigen.

Hyperthermia has been reported to induce a dose-dependent reduction in the expression of melanoma-associated surface antigens. The objective of the present work was to study the mechanisms for the reduction in the expression of the p250 antigen recognized by the monoclonal antibody 9.2.27. Measurements at 37 degrees C showed that antibody binding induced a certain degree of modulation (internalization) of the melanoma-associated antigen. Masking of the antigen due to internalization and/or damage in situ, as well as shedding of the antigen, were measured after hyperthermia, and found to increase in a heat-dose-dependent manner, although for antigen masked this increase was not significant compared with control cells at 37 degrees C. The sum of antigen shed and masked after hyperthermia correlated with the overall reduction in antigen expression measured independently. During hyperthermia, antigen was shed and masked in approximately equal amounts. After the treatment, hyperthermia-induced shedding continued as a function of time and caused a further reduction in antigen expression, but masking did not differ from 37 degrees C controls.

Single cell studies on the immunological marker profile of plasmacytoid T-zone cells.

Plasmacytoid T cells (PTC) are known to home to thymic (T) zones in human lymph nodes and are characterized by their abundant, concentrically layered, rough endoplasmic reticulum. These cells have been found in reactive and neoplastic conditions. Three cases of PTC lymphomas have so far been reported. All of them were complicated by a myelomonocytic leukemia leading to the assumption of a functional relationship between PTC and the myeloid system. The immunologic phenotype of PTC, as revealed on frozen tumor tissue sections, comprised the expression of CD5 (T1), CD4 (T4), and HLA-DR, but not CD8 (T8) and CD2 (T11) and suggested an affiliation to the T cell system. Extending our previous report on one of these cases we here present the first study on the immunological marker profile of suspended PTC. The employment of unfractionated or PTC-enriched tumor cell suspensions rendered possible the application of a panel of monoclonal antibodies (moAbs) on both fixed and unfixed cells and enabled us to allocate various markers either to the intracytoplasmic or surface domain of this cell type. Our results suggest that PTC from our case rest in the G0/G1 phase of the cell cycle. They express the transferrin receptor, but not the Il-2 receptor (CD25) or the nuclear antigen Ki-67. No T cell antigen was demonstrated on the surface of unfixed suspended PTC. Under these conditions only HLA-DR and a predominantly monocytic antigen (CD36/moAb 5F1) were identified. Fixed cells, however, showed a weak cytoplasmic reactivity for CD5 and two myelomonocytic antigens (CD15/moAb 1G10 and CD14/moAb My4). Our findings do not sustain positive evidence for a T cell nature of PTC. Whether their phenotypical pattern indicates terminal differentiation with concomitant loss of T cell antigens or points to a cytogenetic relationship of PTC to the myeloid system, remains speculative. Until the cytogenesis of PTC is clarified we propose the noncommitted term "plasmacytoid T-zone cells" for this elusive cell type.

Selection of anti-SCLC antibodies for diagnosis of bone marrow metastasis.

The binding profile of 17 monoclonal antibodies (MAbs) to small lung cancer cell lines and normal bone marrow and peripheral blood cells was studied by immunocytochemistry and flow cytometry. At antibody concentrations that stained PJD tumour cells, only four MAbs (MOC1, MOC31, NrLu10, 81A6) were devoid of cross-reactivity with normal cells, whereas significant binding to subtypes of bone marrow and blood cells was seen for 13 antibodies. For the eight most promising MAbs the binding to ten SCLC cell lines was moderate to strong in 47 MAb/cell line combinations, and low or insignificantly in 33 combinations. Three cell lines lacked antigen for all MAbs studied. Flow cytometry was significantly less sensitive than immunocytochemistry in assessing MAb binding to both normal and tumour cells. The antigen expression was for several MAbs higher in exponentially growing tumour cells than in cells in stationary growth phase. Seven of the MAbs, which originally showed low to moderate cross-reactivity with normal cells, were titrated down to the lowest concentrations at which they stained H-146 tumour cells with high levels of antigen expression. At these concentrations five (MLuC1, Oat-1, SM-1, NCC-Lu-243, LAM2) of the seven Mabs showed acceptably low binding to bone marrow cells. At optimal concentrations altogether four to nine of the 17 antibodies studied may be used to detect tumour cell involvement in bone marrow of SCLC patients.

Changes in the expression of idiotype antigen on murine B-cell lymphoma after hyperthermia alone and in combination with interferon and tumour necrosis factor.

The effect of interferon (IFN) and tumour necrosis factor (TNF), either alone or combined with hyperthermia, on cell proliferation and expression of idiotype antigen on a murine B-cell lymphoma has been studied. Incubation with same doses of IFN-alpha and IFN-gamma reduced cell proliferation to the same extent. Hyperthermia potentiated the antiproliferative activity of IFN-alpha and IFN-gamma. Pretreatment with IFN-gamma induced a synergistic response with heat, while IFN-alpha and heat had an additive effect. Tumour necrosis factor (TNF) alone did not affect cell proliferation, nor did TNF modify the heat-induced delay in cell growth. The quantitative expression of surface idiotype antigen was studied by flow cytometry using an anti-idiotype monoclonal antibody (MAb). Heat reduced the expression of idiotype antigen approximately 50%. The duration of the reduction depended on the heat-dose. Recovery of antigen expression correlated with recovery of cell growth, and 2-5 days after the treatment antigen expression returned to the normal level for untreated cells. IFN-gamma and TNF increased antigen expression (30-50%) which lasted for 4-6 days after treatment. When cells were incubated with IFN-gamma or TNF for 2 days prior to hyperthermia, the increase in antigen expression was observed immediately after heating, but by the following day, antigen expression was similar to that after heat treatment alone. Expression of idiotype antigen recovered within 2-5 days to the same values as after heat treatment alone. IFN-alpha alone or combined with hyperthermia did not have any significant effect on antigen expression.

Quenching of Hoechst 33258 fluorescence in erythroid precursors.

Quenching of the fluorescence of DNA-bound Hoechst 33258 in erythroid precursors was studied by flow cytometry and cytochemistry. This quenching artifact may affect the measurement of ploidy in specific cases. The bone marrow cells of two patients with hemolytic disease and active erythropoiesis contained subpopulations of cells with an apparent hypodiploid DNA content as measured by flow cytometry of paraformaldehyde-fixed cells stained with Hoechst 33258. No aneuploidy was detected in either of the two cases when cells were stained with mithramycin or 7-aminoactinomycin D. Cells exhibiting reduced Hoechst 33258 fluorescence expressed glycophorin A and low amounts of CD36, and were therefore erythroid precursors. In one case studied, the number of cells with reduced Hoechst 33258 fluorescence and glycophorin A expressed agreed well with the number of cells containing nuclear hemoglobin. In the other case, hemoglobin was present in a significant proportion of nucleated cells. Calculated values for the efficiency of resonance energy transfer from Hoechst 33258 to hemoglobin were in accordance with the observed levels of quenching (approximately 10%). However, the results could also be explained by hemoglobin reabsorption of Hoechst 33258 fluorescence. Nuclei stained with Hoechst 33258 showed uniform fluorescence, probably due to extraction of hemoglobin during the isolation procedure.

The activation-associated antigen 4F2 predicts patient survival in low-grade B-cell lymphomas.

Expression of the activation-associated 4F2 antigen, transferrin receptor and interleukin-2 (IL-2) receptor on suspended cells from 75 biopsied low-grade non-Hodgkin lymphomas (L-NHL) of B-cell origin was correlated to patient survival, clinical prognostic parameters and estimated DNA synthesis. 4F2 antigen expression correlated significantly with poor patient survival, high DNA synthesis and transferrin receptor expression. Transferrin receptor expression was associated with high DNA synthesis and treatment response, but not with patient survival. On the other hand, IL-2 receptor was correlated neither to patient survival nor to other studied markers for cell activation, but seemed to be expressed on certain subsets of lymphomas. We suggest that monoclonal antibody (MAb) against the activation-associated 4F2 antigen could be used to select patients with L-NHL for aggressive chemotherapy.