Thermal sensitivity and thermal tolerance of human B-lineage acute lymphoblastic leukemia (ALL) cells

The thermal sensitivities of four B-cell precursor acute lymphoblastic (ALL) cell lines (REH and KM-3 = pre pre B-ALL; NALM-6 and HPB-NULL = pre B-ALL), and 1 B-cell ALL (NAMALWA) cell line were studied and compared to the thermal sensitivity of the T-lineage ALL cell line MOLT-3 using an in vitro clonogenic assay system by limiting dilution. B-lineage ALL cells were as sensitive to hyperthermia as were T-lineage ALL cells. D0 values at 42 degrees C ranged from 44.9 min (NALM-6) to 85.6 min (NAMALWA), D0 values at 43 degrees C ranged from 15.3 min (NALM-6) to 35.7 min (KM-3), and D0 values at 44 degrees C ranged from 11.1 min (NALM-6) to 23.8 min (HPB-NULL). By comparison, the D0 values of MOLT-3 cells were 95.1 min at 42 degrees C, 23.8 min at 43 degrees C, and 14.7 min at 44 degrees C. The maximum log kill values which were observed ranged from 0.8 log (KM-3 and HPB-NULL) to 1.3 logs (NALM-6) at 42 degrees C, from 1.4 logs (KM-3) to 4.2 logs (NALM-6) at 43 degrees C, and from 3.8 logs (HPB-NULL) to 4.8 logs (NALM-6) at 44 degrees C. A thermal tolerant plateau was observed in the hyperthermia survival curves of REH, NALM-6, and HPB-NULL cells, providing circumstantial evidence that thermal tolerance may develop in some B-cell precursor ALL cells after 90-120 min of continuous heating. In contrast, no thermal tolerant plateau was observed in the hyperthermia survival curves of pre-pre-B-ALL/KM-3 B-cell ALL/NAMALWA or T-lineage ALL/MOLT-3 cells. The kinetics of development and decay of thermotolerance was studied for NALM-6 cells. Thermotolerance after a priming heat exposure to 42 degrees C for 30 min was maximum at 8 hr with a maximum thermotolerance ratio of 2.0, and it decayed by 24 hr. These findings extend previous studies on the thermal sensitivity of human leukemia cells and provide new information on the thermal sensitivity and thermotolerance of B-lineage ALL cells.     

Radiation reduces pirarubicin sensitivity in human cervical squamous cell carcinoma cells

To find an effective protocol for chemoradiotherapy with pirarubicin hydrochloride (THP) for advanced cervical cancer patients, effects of irradiation on THP sensitivity were examined using the radiosensitive human cervical squamous cell carcinoma cell line ME180. Concurrent irradiation significantly reduced THP sensitivity, and this was further reduced when cells were treated with THP 8 h after irradiation. However, the THP sensitivity of cells treated with THP 8 h before irradiation was significantly enhanced. Four months after the first irradiation, 4 subclones of ME180 cells that survived repetitive irradiation demonstrated significantly higher THP sensitivity than non-irradiated parent cells. These results indicate that THP injections, more than several hours before irradiation or after completion of radiotherapy, might be better therapies than concurrent chemoradiotherapy with THP for cervical cancer.     

Radiation sensitivity in vitro of cells isolated from human tumor surgical specimens

The radiation sensitivity of cells isolated directly from human tumor surgical specimens was studied using the Courtenay soft agar colony assay. Aerobic cell survival curves covering 2-3 decades were achieved for eight melanomas, seven ovarian, six cervix, five breast, four bladder, and four squamous cell carcinomas of the head and neck and two seminomas. Cell survival following exposure to 2.0 Gy was measured also for several other tumors of these histological types. Experiments repeated with cells stored in liquid nitrogen showed that the survival assay gave highly reproducible results. The D0 (0.61-1.65 Gy) as well as the surviving fraction at 2.0 Gy (0.12-0.66) differed considerably among individual tumors of the same histological type. Neither of these parameters was therefore significantly different for the seven tumor categories. However, about one-third of the melanomas showed a higher surviving fraction at 2.0 Gy than the highest value measured for the other tumors. Two of three seminomas showed surviving fractions at 2.0 Gy in the absolute lower range, i.e., below 0.20. Altogether the data were consistent with the suggestion recently put forward that the clinical radiocurability of tumors may be correlated to the cell surviving fraction in vitro at 2.0 Gy. However, it was not possible on the basis of individual tumors to investigate whether the surviving fraction at 2.0 Gy was correlated to the clinical radiocurability, since adequate clinical data were not available for the parent tumors. It is suggested that melanomas may be especially suitable for prospective studies aimed at establishing whether such a correlation really does exist. If a significant correlation can be verified, then a very important conclusion may be drawn from our data: the radiocurability of human tumors may differ almost just as much among individual tumors of the same histological type as among individual tumors of different histology.     

Radiation and heat sensitivity of cells from two slowly growing human melanoma xenografts

The radiation and heat sensitivity of cells from two melanin-rich, slowly growing human melanoma xenografts (B.E. and R.A.) were studied. The volume-doubling times of the xenografts in the volume range 200-500 mm3 were found to be 22.5 47.5 days (B.E.) and 25.3-39.2 days (R.A.). The cells were suspended in culture medium during irradiation or heating, and the colony forming ability of the cells was assayed in soft agar. The X-ray survival curve parameters were found to be: Do = 1.09 +/- 0.14 Gy, Dq = 1.99 +/- 0.58 Gy (B.E.); Do = 1.23 +/- 0.08 Gy, Dq = 2.03 +/- 0.35 Gy (R.A.). The Do-values of the heat survival curves were found to be 119.0 +/- 26.6 min (42.5 degrees C), 20.4 +/- 3.9 min (43.5 degrees C) and 9.6 +/- 1.6 min (44.5 degrees C) for the B.E. melanoma and 112.9 +/- 13.3 min (42.5 degrees C), 17.9 +/- 2.0 min (43.5 degrees C) and 7.7 +/- 0.5 min (44.5 degrees C) for the R.A. melanoma. Both the radiation and the heat sensitivities of the cells are within the range of sensitivities reported for rapidly growing melanoma xenografts, suggesting that the intrinsic radiation and heat sensitivity of tumour cells are not strongly related to the rate of tumour growth prior to treatment.     

Radiation sensitivity of cultured rabbit aortic endothelial cells

The radiation response of cultured rabbit aortic endothelial cells was measured using colony formation to determine survival. The dose survival curve was qualitatively similar to those reported for other cell types. At doses of 400 rad and greater, the curve was an exponential function of dose with a D0 of 120 rad and an n of 7. Exponentially growing endothelial cell cultures recovered from sublethal damage between two doses of radiation. Plateau phase cultures recovered from potentially lethal damage when incubated for 6 hr between irradiation and assay.     

Radiation sensitivity of leukemic progenitor cells in acute nonlymphocytic leukemia

The radiation sensitivity of leukemic progenitor cells in 12 cases of acute nonlymphocytic leukemia was compared with that of normal myeloid progenitor cells (colony-forming units in culture), using in vitro cloning techniques. The D0 value for normal colony-forming units in culture was almost constant (130 +/- 14 rads). On the other hand, marked patient-to-patient variations were observed in the radiosensitivity of leukemic progenitor cells; namely, the D0 values in the present cases ranged from 30 to 210 rads. These variations seemed to be partly due to different cell cycle status and partly due to the intrinsic nature of leukemic progenitor cells. Moreover, in six of seven clinically drug-sensitive cases, the leukemic progenitor cells were proved to be relatively radiosensitive. Similar to in vitro drug sensitivity tests, this test may serve to predict the clinical response to chemoradiotherapy.     

Radiation repair in human endothelial cells

Damage to blood vessels caused by ionizing radiation is considered to be an important dose limiting factor for late effects in many organs. However, the radiation response of the endothelial cells which line the vasculature has not been well-documented, particularly for human endothelial cells. In the present study, human endothelial cells were obtained from fresh umbilical cords and cultured in monolayer. Immunofluorescent staining of factor VIII antigen was used to verify the endothelial nature of the cultured cells. The cells were irradiated with Cs-137 rays (1.35 Gy/min) in plateau phase to determine radiation sensitivity and ability to repair potentially lethal damage (PLD) and sub-lethal damage (SLD). The endothelial cells demonstrated moderate radiosensitivity that varied slightly between different cords. As demonstrated by delayed plating experiments, PLD repair ability was substantial, with repair factors of 0.70-0.80. SLD repair capability, as demonstrated by split dose experiments, was relatively modest. A survival enhancement of 2.0-2.2, for example, was observed when 8 Gy single dose survival was compared to two 4 Gy doses. In terms of PLD and SLD repair as well as initial slope (alpha) the endothelial cells were similar to normal human lung and skin fibroblasts previously studied. Compared to malignant cell lines, PLD repair was generally larger whereas the initial slope (alpha) was intermediate-steeper than the radioresistant tumor types but shallower than the more sensitive tumor derived cells.     

Binding of 125I-labelled human alpha interferon to human lymphoid cells

To investigate the binding of interferon to human lymphoid cells, we purified human alpha interferon and radio-labelled it with iodine-125. Binding at 4 degrees C could be saturated and was inhibited by unlabelled interferon; it was specific for cells of human origin. Dissociation constants for the complex of interferon and receptor site were of the order 10(-9)-10(-11) M. All human cells tested showed such binding. Occupation of these high-affinity sites, at 37 degrees C, was compared with the inhibition of cellular growth due to interferon. The most sensitive cell line (Daudi) gave a complete biological response with only a fraction of its sites occupied. Evidence of two sites was found for a line (P3HRI) showing intermediate sensitivity. A relatively insensitive line (Raji) showed no response when all its high-affinity sites were occupied.     

Specific uptake of alpha-fetoprotein by malignant human lymphoid cells

We have studied the ability of human B- and T-lymphoblastoid cell lines, as well as of peripheral lymphoid cells from leukemia patients, to take up alpha-fetoprotein (AFP) and other serum proteins. Two technical approaches have been employed, both using fluorescent protein derivatives (FITC-proteins): microscopic examination of labelled cells using epifluorescent illumination and quantitation of endocytosed proteins by fluorescence-activated cell sorting (FACS). Compared to human resting T-lymphocytes, all T- and B-cell lines tested exhibited positive staining for fluoresceinated AFP and transferrin (Tf) and a significant increase, up to 100-fold, of the number of AFP and Tf molecules endocytosed per cell. Labelling was prevented or strongly diminished by a 100-fold excess of unlabelled protein. Preliminary results with peripheral lymphoid cells harvested from leukemia patients showed the presence of AFP- and Tf-positive cells in the blood of all patients examined. Intensity of labelling was related to the type of leukemia cells and/or the degree of cell maturation. Most cell lines exhibited positive staining for alpha 2-macroglobulin (alpha 2M) and also, to a lesser extent, for serum Vitamin D3 binding protein (DBP). In contrast, no labelling was observed with FITC-serum albumin (FITC-Alb) or FITC-ovalbumin (FITC-OVA). Comparative uptake of several FITC-proteins by a single cell population revealed significant quantitative and qualitative differences.     

Circulating myeloid and lymphoid precursor dendritic cells are clonally involved in myelodysplastic syndromes.

Circulating myeloid and lymphoid precursor dendritic cell (pDC) counts were determined in peripheral blood from 22 patients with myelodysplastic syndromes (MDS) by a single-platform flow cytometric protocol. The absolute count of myeloid and lymphoid pDC, as well as their relative number (as proportion of mononuclear cells or total leukocytes) was significantly lower in MDS (n=22) than in healthy controls (n=41). In 11 patients with chromosomal aberrations, purified pDC were examined by interphase fluorescence in situ hybridization. This revealed clonal involvement of myeloid as well as lymphoid pDC in all of them. These data therefore strongly suggest that myeloid and lymphoid pDC share a common precursor. Whether reduced peripheral blood counts of pDC contribute to the immunological abnormalities observed in MDS remains to be investigated.     

Radiation and heat sensitivity of cells from human melanoma xenografts. Lack of correlations with tumour growth parameters

Five human malignant melanomas grown in athymic nude mice were studied. Tumour volume-doubling times were determined from Gompertzian growth curves, vascular volumes from stereological analysis of 2-μm thick tumour sections and DNA histograms by flow cytometric analysis. Single-cell suspensions prepared from the tumours were exposed to radiation or heat (42.5°C; pH 7.4) under aerobic conditions in vitro and the colony-forming ability of the cells was assayed in soft agar. Tumours with short volume-doubling times tended to show higher fractions of cells in S-phase and higher vascular volumes than those with long volume-doubling times. The radiation and the heat sensitivity of the melanoma cells, i.e. the D₀-values, were probably not positively correlated with the tumour volume-doubling time, the fraction of cells in S-phase or the vascular volume, or with each other either. The variation in radiation and heat sensitivity among cells from the different melanomas appears not to be due to external factors, but reflects, rather, intrinsic cellular differences.     

Notch-1 and Notch-2 exhibit unique patterns of expression in human B-lineage cells.

The Notch genes encode a conserved family of receptors that influence developmental fate in many species. Prior studies have indicated that Notch-1 and Notch-2 signaling influence the development of hematopoietic stems cells and thymocytes, but little is known regarding Notch expression and function in B-lineage cells. We analyzed the expression of Notch receptors and Notch ligands in human B-lineage cells and bone marrow (BM) stromal cells. Notch-1 mRNA and protein is expressed throughout normal B cell development and in leukemic B-lineage cells. In contrast, Notch-2 expression is limited to pre-B cells expressing low levels of surface mu. The Notch ligand Delta is expressed in BM B-lineage cells. The Notch ligand Jagged-1 is not expressed in B-lineage cells, but is expressed in BM stromal cells. These results suggest a model wherein lateral signaling between Notch and Delta on B-lineage cells and/or Notch/Jagged-1 interactions between B-lineage cells and BM stromal cells may regulate human B cell development.     

Kinetic response to cultured human lymphoid cells to rubidazone

Analysis of rubidazone, the benzoylhydrazone derivative of daunorubicin, for its effects on cell cycle progression of a human lymphoid cell line showed a kinetic response pattern similar to that of adriamycin. Thus rubidazone induced a G2-block, the magnitude and duration of which were dependent on concentration and incubation time. However, in contrast to adriamycin, a marked phase-dependent sensitivity for the induction of G2-accumulation was observed; cells treated in early and mid-S-phase were most sensitive. This age-dependent kinetic response may account for the smaller G2-accumulation in asynchronous cultures and the closer correlation of the magnitude of this kinetic effect with concentration and duration of rubidazone treatment. Prolonged exposure to high concentrations of rubidazone also delayed the traverse through G1 and/or the G1-S transition, whereas the S-phase transit was not impaired. Interference with cell cycle progression through G1 into S-phase caused a stepwise accumulation of cells in G2-phase.     

Effect of tumor necrosis factor on human hematopoietic precursor cells

The effect of human recombinant tumor necrosis factor (rhTNF-alpha) on the in vitro colony growth of normal hematopoietic progenitor cells was investigated. In a clonal colony-assay a dose-dependent inhibition of erythroid BFU-E, granulocyte/monocyte CFU-GM and megakaryocyte CFU-Mk was demonstrated. CFU-Mk were completely inhibited by low doses of TNF-alpha (3 U-300 U/ml). 50% inhibition occurred at 10 U/ml of TNF for CFU-Mk, 100% inhibition at 300 U/ml of TNF. 50% inhibition occurred at 233 U/ml of TNF for CFU-GM, and 100% inhibition for CFU-GM was not observed. For inhibition of BFU-E higher doses of TNF-alpha were necessary (100 U-1,000 U/ml). The growth inhibitory effect could selectively be abolished by antibodies against TNF-alpha. Removal of adherent cells and T-lymphocytes from the bone marrow cells had no significant influence of the suppressive effect of TNF-alpha. The inhibitory effect of TNF-alpha is due to a direct action on hematopoietic progenitor cells and not mediated by accessory cells.     

Radiation therapy of conjunctival and orbital lymphoid tumors

Lymphoid tumors of the conjunctiva and orbit are rare and remain localized in the majority of cases. Sometimes it is not possible either clinically or histologically to differentiate between a non-Hodgkin's lymphoma (NHL) and benign lymphoid hyperplasia. A series of 24 patients is reported. Nineteen were classified as having malignant NHL and 5 benign hyperplasia; 1 of these 5 later developed metastases, however. All patients had systemic work-up: 18 had Stage I, 1 had Stage II, and 5 had Stage IV disease. All patients received local radiation therapy with doses of 2400 to 2750 rad in 2-3 weeks for lesions of the eyelid and conjunctiva, and between 3000 and 3750 rad in 3-4 weeks for retrobulbar lesions. The lens was shielded in all patients except in 2 who had NHL of the vitreous body. A method of shielding the lens with a lead block mounted on a "low vac lens" is described, and the dose distribution within the eye and orbit is presented. The dose to the ocular lens is reduced to about 10% of the tumor dose with this technique. Patients who were treated with doses higher than 3000 rad experienced conjunctivitis and skin erythema that resolved completely. No other effects of radiation on normal structures of the ocular adnexa were observed in the 20 patients who are alive and without signs of tumor 10-46 months with a median follow-up time of 22 months.     

Effect of ionizing radiation on human skeletal muscle precursor cells

Background

Long term effects of different doses of ionizing radiation on human skeletal muscle myoblast proliferation, cytokine signalling and stress response capacity were studied in primary cell cultures.

Materials and methods

Human skeletal muscle myoblasts obtained from muscle biopsies were cultured and irradiated with a Darpac 2000 X-ray unit at doses of 4, 6 and 8 Gy. Acute effects of radiation were studied by interleukin – 6 (IL-6) release and stress response detected by the heat shock protein (HSP) level, while long term effects were followed by proliferation capacity and cell death.

Results

Compared with non-irradiated control and cells treated with inhibitor of cell proliferation Ara C, myoblast proliferation decreased 72 h post-irradiation, this effect was more pronounced with increasing doses. Post-irradiation myoblast survival determined by measurement of released LDH enzyme activity revealed increased activity after exposure to irradiation. The acute response of myoblasts to lower doses of irradiation (4 and 6 Gy) was decreased secretion of constitutive IL-6. Higher doses of irradiation triggered a stress response in myoblasts, determined by increased levels of stress markers (HSPs 27 and 70).

Conclusions

Our results show that myoblasts are sensitive to irradiation in terms of their proliferation capacity and capacity to secret IL-6. Since myoblast proliferation and differentiation are a key stage in muscle regeneration, this effect of irradiation needs to be taken in account, particularly in certain clinical conditions.     

X-ray sensitivity of human tumor cells in vitro

Clonally-derived cells from ten human malignant tumors considered radiocurable (breast, neuroblastoma, medulloblastoma) or non-radiocurable (osteosarcoma, hypernephroma, glioblastoma, melanoma) were studied in cell culture and their in vitro x-ray survival curve parameters determined ($\text{n}$, D_o). There were no significant differences among the tumor cell lines suggesting that survival parameters in vitro do not explain differences in clinical radiocurability. Preliminary investigation with density inhibited human tumor cells indicate that such an approach may yield information regarding inherent cellular differences in radiocurability.     

CDK1 is a marker of proliferation in human lymphoid cells

To better understand the relationship between the proliferation of human lymphoid cells and the expression of cdk1, a catalytic subunit of the histone HI kinase (HIK), we examined its mRNA and protein content in 3 B-cell lines: Ramos, Reh-6 and IARC 963. Cells were elutriated according to their position in the cell cycle. Cell fractions were analyzed for cdk1 mRNA and protein cellular content by Northern blot and immunoblot, respectively, as well as for H1K activity. Both mRNA and protein amounts and H1K activity varied according to cell cycle phase, the lowest values being observed in G₁-enriched fractions. For comparison, elutriated fractions were also tested for the expression of cdk2 and cdk4 proteins. Both showed some variations among fractions, but they were less clear than those of cdk1. We also tested 29 samples of lymphoid neoplastic and non-neoplastic tissues for proliferative activity (percentage of S and G₂/M cells estimated by flow cytometry) and expression of cdk1, cdk2 and cdk4 proteins. We found a significant correlation between the percentage of cells in S or S + G₂/M phases and cdk1 protein content but not cdk2 or cdk4 content. We conclude that cdk1 expression in human lymphoid cells varies during the cell cycle at both mRNA and protein levels. © 1995 Wiley-Liss, Inc.