Enhancement of murine bone marrow ablation by combining whole body hyperthermia with body irradiation and cyclophosphamide
Bone marrow ablation using combined whole body hyperthermia (WBH), total body irradiation (TBI), and cyclophosphamide (Cy) was investigated in C3H f/Sed mice to demonstrate cytotoxic synergism between the three modalities. TBI was given on day 0. WBH treatment was for 1 hr at 41.8 degrees C, given in daily sessions for 1, 2 or 3 modalities. TBI was given on day 0. WBH treatment was for 1 hr at 41.8 degrees C, given in daily sessions for 1, 2 or 3 consecutive days following TBI. Total cyclophosphamide doses were 160 and 240 mg/kg given in 2 daily injections on days 1 and 2 following TBI. Polymorphonuclear leukocyte and lymphocyte numbers were determined by differential cell counts. The total peripheral blood cell counts were also determined. WBH alone, given in daily sessions for 3 days, did not reduce the total peripheral blood cell counts. However, when WBH was added to TBI (6.3 Gy) peripheral blood cellularity was reduced on day 2, but no significant heat/radiosensitization was evident after day 2. WBH (3 daily sessions) significantly reduced the peripheral blood cellularity and resulted in bone marrow ablation when it was combined with TBI and Cy. CY (160-240 mg/kg) combined with TBI (5.4 Gy) resulted in bone marrow ablation and subsequent death in 14-22% of mice treated; 60-100% of mice died from bone marrow ablation when WBH was added to TBI (5.4 Gy) and Cy (160-240 mg/kg). Femoral and vertebral tissue sections showed total loss of progenitor cells when WBH, TBI (5.4 Gy), and Cy (240 mg/kg) were combined whereas lessor treatment was associated with histologically verified reconstitution of progenitor cells inside the marrow cavities. These studies indicate that bone marrow ablation can be achieved when using WBH in combination with lower doses of TBI and Cy.
Available from: Hayato Miyachi
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ABSTRACT: In these studies, heat radiosensitization in normal human colony-forming unit-granulocyte-macrophage (CFU-GM) and several different leukemic cell lines sensitive or resistant to chemotherapeutic agents were measured. Extent of heat radiosensitization was then correlated with the level of DNA polymerases alpha and beta in control and heat-shocked cells in order to examine whether there is a positive correlation between the degree of heat radiosensitization and the level of these enzymes. Our results show that human bone marrow CFU-GM have an x-ray response with D0 of 1.56 Gy and a small amount of heat radiosensitization with a thermal enhancement ratio (TER) of 1.2. K562, a human erythroleukemic cell, showed a D0 of 1.32 +/- 0.2 Gy and TER of 1.4. However, in contrast to normal CFU-GM which showed no shoulder in the X-ray survival curve, K562 cells showed a small shoulder with a quasi-threshold dose, (Dq) of 2 Gy and n of 2. K562 cells resistant to chemotherapeutic drugs such as 1-beta-D-arabinofuranosylcytosine and etoposide (VP-16) showed D0 of 1.47 +/- 0.13, and 1.77 +/- 0.18 Gy; Dq of 4 and 0 Gy; and n of 5 and 1; and TER of 1.6 and 2, respectively. The level of DNA polymerases alpha and beta activity and their respective mRNA levels were approximately the same in all cells. The reduction in the level of DNA polymerase beta after heat treatment however, correlated with the TER obtained for various leukemic cells. These studies indicate that normal CFU-GM and variety of human leukemic cells show only a small amount of heat radiosensitization. However, drug-resistant leukemic cells show a higher amount of heat radiosensitization than their drug-sensitive parent line. This suggests that hyperthermia may be beneficial in eradicating drug-resistant leukemic cells when combined with X-ray. Furthermore, the inactivation of DNA polymerase beta activity results in a higher amount of heat radiosensitization.
Available from: cancerres.aacrjournals.org
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ABSTRACT: The expression of the Mr 70,000 heat shock protein (HSP-70) in heat-resistant variants or heat-shocked cells has been correlated with development of thermal resistance. In these studies polymerase chain reaction (PCR) was used to detect low levels of HSP-70 mRNA present in control, unheated cells to investigate the possibility of predicting the intrinsic heat response in various leukemic cells. The expression of two human heat shock genes in control or heat-shocked cells was investigated. Synthetic primers and probes from the untranslated region of the two HSP-70 genes sequenced by Hunt and Morimoto (HSP-70A)(C. Hunt and R. I. Morimoto, Proc. Natl. Acad. Sci. USA, 82: 6455-6459, 1985) and Voellmy et al. (HSP-70B)(R. Voellmy et al., Proc. Natl. Acad. Sci. USA, 82: 4949-4953, 1985) were used in PCR reactions to follow expression in control or heat-shocked leukemic K562, KG-1, and HL-60 cells. The PCR results were correlated with heat response and patterns of protein synthesis in these cells. Results indicate that, among leukemic cells, K562 was much more resistant to killing by heat shock than either KG-1 or HL-60 cells. All control cells, however, expressed the HSP-70B gene. Of the three leukemic cells tested, K562 was the most heat resistant and constitutively expressed the HSP-70A mRNA and the heat-inducible HSP-70 protein. KG-1 and HL-60 cells did not express this gene in unheated cells. All heat-shocked cells expressed the HSP-70A mRNA and the heat-inducible HSP-70 protein. However, there was no significant increase in the mRNA level of the HSP-70B in heat-shocked leukemic cells as measured by PCR or the S1-nuclease protection assay. Other cells including normal human bone marrow and normal and tumorous tissues of the colon and breast all expressed both genes in control cells. Normal breast tissue expressed less mRNA for HSP-70B gene than the tumor tissue obtained from the same patient. In all studies the amplified beta-actin mRNA expression was used as an internal standard. These studies indicate that HSP-70B gene is expressed in all control leukemic cells. The expression of this gene did not seem to correlate with intrinsic heat resistance. The HSP-70A expression correlated with intrinsic and transient heat resistance. These studies also indicate that both HSP-70 genes in humans may be expressed in a variety of unheated normal and tumorous tissues more so than previously reported.
Available from: dissertations.ub.rug.nl
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ABSTRACT: The hyperthermic sensitivity of the CFU-S-12 in bone marrow from normal and anaemic mice was determined. The terminal slope of the survival curves, demonstrated by the T0 values, does not significantly differ in the resting and active cycling stem cells. In the active cycling stem cells the initial shoulder region was less dominant compared with the resting stem cells. The difference in heat sensitivity between resting and active proliferating CFU-S-12 might be explained by a difference in the accumulation of damage before lethality becomes manifest. The difference in heat sensitivity appears to be independent of the environmental accessory cells, demonstrated by a similar hyperthermic effect of the purified stem cells from bone marrow and spleen and the stem cells in the total cell suspensions. Therefore the heat sensitivity of the haemopoietic stem cell is not mediated by a release of injurious substances from environmental heat-damaged cells. The heat treatment does not result in a selection of macroscopic detectable colonies 12 days after inoculation, as is demonstrated by the same morphology of the spleen colonies from the stem cells before and after the hyperthermic treatment.
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