Biomedical subjects

D B Leeper

Publications and source records attributed to D B Leeper.

At least 19 recordsLinked to original sources

PubMed2004

Acute extracellular acidification reduces intracellular pH, 42 degrees C-induction of heat shock proteins and clonal survival of human melanoma cells grown at pH 6.7.

Two human melanoma cell lines, SK-Mel-28 and DB-1, were used for in vitro studies of the mechanisms underlying heat resistance of human tumour cells adapted to growth in acidic environments. Adaptation to growth at low pH was characterized by resistance to 42 degrees C cytotoxicity and accompanied by an increase in endogenous levels of Hsp70 and/or Hsp27. Acute extracellular acidification to levels below pH 6.5 was required to sensitize the melanoma cells to 42 degrees C. Furthermore, cells grown at low pH were more resistant to sensitization by acute acidification than cells grown at pH 7.3. The intracellular pH (pHi) of cells grown at pH 6.7 was less than the pHi of cells grown at pH 7.3 both before and after acute acidification. A pHi threshold existed for melanoma cells growing at pH 7.3 below which they became sensitized to 42 degrees C. This pHi threshold differed between the SK-Mel-28 and DB-1 cells. In contrast, a pHi threshold for heat sensitization did not exist for cells growing at pH 6.7: any reduction in pHi before heating resulted in increased cell killing. Since cells grown at low pH lack a pHi threshold for heat sensitization, they are sensitized more to 42 degrees C per unit decrease in pHi than cells grown at pH 7.3. Acute acidification abrogated the 42 degrees C-induction of Hsp70 and Hsp27 in the melanoma cells. The pHi thresholds for abrogation of these HSPs are slightly higher than or comparable with the thresholds for cytoxicity for each cell line grown at pH 7.3, but abrogation occurred over a narrower range of pHi compared with cytotoxicity. Abrogation of heat-induced expression of these HSPs correlates with cytotoxicity in both cell lines with the exception of Hsp27 expression in SK-Mel-28 cells. In conclusion, strategies that reduce pHi in melanoma cells growing at low pH, such as in acidotic regions of tumours, could selectively sensitize them to hyperthermia because they lack a pHi threshold for heat sensitization.

Blotting, Western↗

PubMed2004

Feasibility of lung cancer hyperthermia using breathable perfluorochemical (PFC) liquids. Part II: Ultrasound hyperthermia.

Enhanced local control of disease in lung cancer has been shown to improve survival, and controlled clinical trials of hyperthermia adjunctive to radiotherapy in other cancers have shown improved disease control and survival over radiotherapy alone. The challenge of lung hyperthermia, however, persists. This investigation sought to demonstrate the feasibility of localized lung hyperthermia at depth via therapeutic ultrasound. The method is based on using breathable perfluorochemical liquids as acoustic coupling media in the lung, liquids that have also been shown to enable controlled liquid-filled lung convective hyperthermia (LCHT). The ability to use both lung convective hyperthermia and liquid-filled lung ultrasound hyperthermia (LUHT) provides potential flexibility in heating patterns for the hyperthermic treatment of lung cancer with concurrent radiotherapy and/or chemotherapy. Using custom ultrasound transducers designed and built for these studies, the acoustic properties of three candidate perfluorochemicals were characterized over a range of temperatures, gas contents and ultrasound frequencies and acoustic intensities. Both sound speed and attenuation were measured in the neat liquids and in isolated lungs filled with the perfluorochemicals. Successful ultrasound hyperthermia at depth was demonstrated in vivo in sheep lung lobes in intraoperative conditions. In addition, the use of ultrasound diagnostic imaging was explored as a tool for use in conjunction with lung ultrasound hyperthermia.

Acoustics↗

PubMed2004

Feasibility of lung cancer hyperthermia using breathable perfluorochemical (PFC) liquids. Part I: Convective hyperthermia.

Clinical studies have shown that hyperthermia in combination with radiotherapy and/or chemotherapy may be effective in the treatment of advanced cancer. No method of lung hyperthermia, however, has been accepted as standard or superior. This investigation sought to demonstrate in animals the thermal and physiologic feasibility of lung hyperthermia induced using heated breathable perfluorochemical (PFC) liquids, a method termed liquid-filled lung convective hyperthermia (LCHT). The ability to use LCHT is rooted in the development of both PFC liquid ventilation, now in clinical development with the PFC perflubron (LiquiVent), and a PFC blood substitute also in late Phase III trials (Oxygent). As LCHT background, the PFC technologies and biology are first reviewed. The physical properties of a variety of PFCs were evaluated for LCHT and it was concluded that more than one liquid is suitable based on such properties. Using total liquid ventilation type devices, LCHT was shown to deliver successfully localized (lobar) lung heating in sheep, and bilateral whole lung heating and whole-body hyperthermia in rabbits, cats and lambs. During LCHT, lung parenchymal temperatures were uniform (<1 degree C) across heated regions. In addition, based on patterns relating lung tissue temperatures to inspiratory and expiratory PFC liquid temperatures in the endotracheal tube, LCHT may minimize invasive thermometry requirements in the lung. Based on acute experiments, it was concluded that LCHT appears feasible and may simplify lung hyperthermia. It was recommended that potentially synergistic combinations of LCHT with other whole-body hyperthermia or local heating modalities, and with chemotherapeutic lung drug delivery, also be explored in the future.

Animals↗

PubMed2004

Hsp27 anti-sense oligonucleotides sensitize the microtubular cytoskeleton of Chinese hamster ovary cells grown at low pH to 42 degrees C-induced reorganization.

Chinese hamster ovary (CHO) cells maintained in vitro at pH 6.7 were used to model cells in the acidic environment of tumours. CHO cells grown at pH 6.7 develop thermotolerance during 42 degrees C heating at pH 6.7 and their cytoskeletal systems are resistant to 42 degrees C-induced perinuclear collapse. Hsp27 levels are elevated in cells grown at pH 6.7 and are further induced during 42 degrees C heating, while Hsp70 levels remain low or undetectable, suggesting that Hsp27 is responsible for some of the novel characteristics of these cells. An anti-sense oligonucleotide strategy was used to test the importance of Hsp27 by lowering heat-induced levels of the protein. The response of the microtubular cytoskeleton to heat was used as an endpoint to assess the effectiveness of the anti-sense strategy. Treatment with anti-sense oligonucleotides prevented the heat-induced increase of Hsp27 levels measured immediately following heat. Treatment with anti-sense oligonucleotides also sensitized the cytoskeleton of cells grown at low pH to heat-induced perinuclear collapse. However, cytoskeletal collapse was not evident in cells grown at pH 6.7 and treated with 4-nt mismatch oligonucleotides or in control cells maintained and heated at pH 6.7. The cytoskeleton collapsed around the nucleus in cells cultured and heated at pH 7.3. These results confirm that over-expression of Hsp27 confers heat protection to the microtubular cytoskeleton in CHO cells grown at low pH.

Animals↗

PubMed2002

Variability in glucose transporter-1 levels and hexokinase activity in human melanoma.

Melanoma exhibits heterogeneous growth patterns and widely varying sensitivities to multiple treatment modalities. This variability may reflect intrinsic genetic differences in factors giving rise to altered metabolism. Glucose is the primary energy source of tumours, including melanoma, and glucose transporter isoform 1 (Glut-1) and hexokinase are key rate-limiting factors in glucose metabolism. The levels of Glut-1 and total hexokinase activity were measured in 31 melanoma biopsies to determine the extent of tumour-to-tumour variability in these parameters. Relative Glut-1 levels were determined by Western immunoblot analysis using human anti-Glut-1 rabbit polyclonal antibody, and hexokinase activity was measured in the same samples by an enzymatic assay monitoring the reduction in the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) (in nmol NADP+ reduced/min per mg protein). All melanomas were from patients who had received no therapy prior to surgery. Immediately after excision, tumour biopsies were disaggregated to single cells by collagenase and DNase and frozen in liquid nitrogen. Thirty human melanomas exhibited a 22-fold variation in levels of Glut-1 and 29 exhibited a nine-fold variation in total cellular hexokinase activity. Glut-1 levels and hexokinase activity were not correlated with one another. The broad range in Glut-1 levels and hexokinase activity observed between melanomas suggests that these glycolytic rate-limiting parameters that influence the rate of glucose metabolism may contribute to the variability in melanoma response to treatment modalities.

Adult↗

PubMed2001

Absence of Crabtree effect in human melanoma cells adapted to growth at low pH: reversal by respiratory inhibitors.

Because many tumors are acidic and hypoxic relative to normal tissues, glycolysis and oxygen consumption were investigated in early-passage human melanoma cells adapted to growth at pH 6.7. In the absence of glucose, the basal rate of oxygen consumption in low pH-adapted cells was 75% of that in cells grown at pH 7.3. The rate of lactic acid production in low pH-adapted cells was increased 4-fold by exposure to 16.7 mM glucose compared with a 10-fold increase in cells grown at pH 7.3. Furthermore, in low pH-adapted cells the rate of oxygen consumption was stimulated by the addition of glucose in contrast to the inhibition of oxygen consumption by elevated glucose in cells grown at pH 7.3 (i.e., the Crabtree effect). Both low pH-adapted cells and cells grown at pH 7.3 exposed to glucose plus 0.35 mM meta-iodo-benzylguanidine (MIBG), an inhibitor of mitochondrial respiration, had oxygen consumption reduced by approximately 60% and lactic acid production increased by approximately 65% relative to glucose alone. Although adaptation to growth at low pH was associated with a loss of the Crabtree effect and a higher ratio of oxygen consumption to lactic acid production, the rate of glycolysis was the same in both growth conditions in the presence of 0.1 mM dinitrophenol, an uncoupler of ATP synthesis. This indicates that the glycolytic capacity of low pH-adapted cells remains unchanged. Therefore, tumor acute acidification and oxygenation can be achieved by exposure to hyperglycemia combined with MIBG to improve therapeutic response.

3-Iodobenzylguanidine↗

PubMed2001

Enhancement of hyperglycemia-induced acidification of human melanoma xenografts with inhibitors of respiration and ion transport.

RATIONALE AND OBJECTIVES: The authors performed this study to evaluate the selective acidification of a human melanoma xenograft in mice with severe combined immunodeficiency with the induction of hyperglycemia (mean blood glucose level +/- standard error of the mean, 26 mmol/L +/- 1) and the intraperitoneal administration of metaiodobenzylguanidine (MIBG, 30 mg/kg), alpha-cyano-4-hydroxycinnamate (CNCn, 300 mg/kg), lonidamine (100 mg/kg), cariporide (HOE642, 160 mg/kg), or 4.4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS, 50 mg/kg). MATERIALS AND METHODS: The intra- and extracellular pH levels of tumor were estimated from the chemical shifts of inorganic phosphate and 3-aminopropylphosphonate, respectively, with phosphorus-31 nuclear magnetic resonance (MR) spectroscopy. The relative level of steady-state lactate was monitored with hydrogen-1 MR spectroscopy. RESULTS: In small tumors (< or = 8.0 mm), hyperglycemia decreased the intra- and extracellular pH levels by less than 0.2. The combination of hyperglycemia and MIBG decreased the intra- and extracellular pH levels by approximately 0.4 and 0.6, respectively, and lowered the beta-nucleoside triphosphate (NTP)/inorganic phosphate (Pi) ratio of tumor and liver by about 60% and 25%, respectively. The combination of hyperglycemia, MIBG, and CNCn produced a transient decrease in the intracellular pH of about 0.6. The combination of hyperglycemia and lonidamine produced a sustained (>3 hours) 0.8-unit decrease in intracellular pH and an 83% and 100% decrease in PCr/P1 and beta-NTP/P1 ratios, respectively. The combination of hyperglycemia. MIBG, cariporide, and DIDS produced a gradual decrease in intra- and extracellular pH by 1.1 and 1.0, respectively. The relative level of steady-state lactate concentration in tumors increased 10% with hyperglycemia alone, about 20% with MIBG plus hyperglycemia, and increased more than twofold when hyperglycemia was combined with MIBG and CNCn administration. CONCLUSION: These preliminary data suggest that hyperglycemia and combinations of respiratory and ion transport inhibitors can be used to selectively acidify tumors and, thereby, sensitize them to hyperthermnia or other pH-sensitive therapeutic modalities.

3-Iodobenzylguanidine↗

PubMed2000

Intracellular acidification of human melanoma xenografts by the respiratory inhibitor m-iodobenzylguanidine plus hyperglycemia: a 31P magnetic resonance spectroscopy study.

In vivo 31P magnetic resonance spectroscopy demonstrates that human melanoma xenografts can be significantly acidified by induction of hyperglycemia combined with administration of m-iodobenzylguanidine (MIBG), an inhibitor of mitochondrial respiration. In melanoma xenografts (< or =8 mm diameter), intracellular pH (pHi, measured by the chemical shift of the Pi resonance) and extracellular pH (pHe, measured with 3-aminopropylphosphonate) was reduced by less than 0.2 unit during i.v. infusion of glucose for 40 min. Administration of MIBG (30 mg/kg) under hyperglycemic conditions (26 mM) reduced tumor pHi and pHe by approximately 0.4 (P < 0.001) and approximately 0.6 (P < 0.001) unit, respectively; coincidentally, the nucleoside triphosphates:Pi ratio decreased approximately 60% (P < 0.004) relative to the baseline level. Minimal changes in pHi and pHe and a small decrease in nucleoside triphosphates:Pi ratio (26%, P = 0.2) were observed in liver in response to MIBG plus hyperglycemia. These results suggest that under normoglycemic and hyperglycemic conditions, small human melanoma xenografts (< or =8 mm) may exhibit a relatively high level of oxidative phosphorylation that may be blocked by MIBG. The acidification may result from increased lactate production as a direct effect of MIBG inhibition of respiration in mitochondria of tumor cells, or through indirect systemic effects, which remain to be identified. The synergetic effects of MIBG and hyperglycemia result in significant acidification of the tumor and a decrease in tumor bioenergetic status, and the effects are largely selective for tumors in comparison with normal tissues.

3-Iodobenzylguanidine↗

PubMed2000

Intracellular pH regulation in a nonmalignant and a derived malignant human breast cell line.

Tumor cells in vivo often exist in an ischemic microenvironment that would compromise the growth of normal cells. To minimize intracellular acidification under these conditions, these cells are thought to upregulate H(+) transport mechanisms and/or slow the rate at which metabolic processes generate intracellular protons. Proton extrusion has been compared under identical conditions in two closely related human breast cell lines: nonmalignant but immortalized HMT-3522/S1 and malignant HMT-3522/T4-2 cells derived from them. Only the latter were capable of tumor formation in host animals or long-term growth in a low-pH medium designed to mimic conditions in many solid tumors. However, detailed study of the dynamics of proton extrusion in the two cell lines revealed no significant differences. Thus, even though the ability to upregulate proton extrusion in a low pH environment (pH(e)) may be important for cell survival in a tumor, this ability is not acquired along with the capacity to form solid tumors and is not unique to the transformed cell. This conclusion was based on fluorescence measurements of intracellular pH (pH(i)) on cells that were plated on extracellular matrix, allowing them to remain adherent to proteins to which they had become attached 24 to 48 h earlier. Proton translocation under conditions of low pH(e) was observed by monitoring pH(i) after exposing cells to an acute acidification of the surrounding medium. Proton translocation at normal pH(e) was measured by monitoring the recovery after introduction of an intracellular proton load by treatment with ammonium chloride. Even in the presence of inhibitors of the three major mechanisms of proton translocation (sodium-proton antiport, bicarbonate transport, and proton-lactate symport) together with acidification of their medium, cells showed only about 0.4 units of reduction in pH(i). This was attributed to a slowing of metabolic proton generation because the inhibitors were shown to be effective when the same cells were given an intracellular acidification.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Isotope selection for permanent prostate implants? An evaluation of 103Pd versus 125I based on radiobiological effectiveness and dosimetry.

Transperineal interstitial permanent prostate brachytherapy (TIPPB) has become an increasingly popular treatment for early-stage/favorable-risk adenocarcinoma of prostate. Within TIPPB, permanent implants often use either (103)Pd (T(1/2) = 17 days) or (125)I (T(1/2) = 60 days). This review compares the radiobiological and treatment planning effectiveness of (103)Pd and (125)I implants by using the linear-quadratic model with recently published data regarding: prostate tumor cell doubling times, T(pot), alpha and alpha/beta, ratio. The tumor potential doubling times (T(pot)) were determined based on recently published proliferation constants (K(p)). The initial slope of the cell radiation dose survival curve, alpha, the terminal slope beta and the alpha/beta ratio were taken from recent published clinical and cellular results. The total dose delivered from each isotope was the dose used clinically, that is, 120 Gy for (103)Pd and 145 Gy for (125)I. Dale's modified linear-quadratic equation was used to estimate the biological effective dose, the cell-surviving fraction, the effective treatment time, and the wasted radiation dose for different values of T(pot). Treatment plans for peripherally loaded implants were compared. The T(pot) reported for organ-confined prostate carcinomas varied from 16 to 67 days. At short T(pot) both isotopes were less effective, but (103)Pd had much less dependence on T(pot) than (125)I. However, at long T(pot) both isotopes produced similar effects. The minimum surviving fraction for exposure to (103)Pd decreased from 1.40 x 10(-4) to 1.31 x 10(-5) as the T(pot) increased from 16 to 67 days. By contrast for exposure to (125)I, the minimum surviving fraction decreased from 3.98 x 10(-3) to 1.98 x 10(-5) over the same range of T(pot). A comparison of treatment plans revealed that (103)Pd plans required more needles and seeds; however, this was a function of seed strength. Both isotopes had similar dose-volume histograms for prostate, urethra, and rectum. The theoretical prediction of effectiveness using the linear quadratic equation for the common clinically prescribed total radiation doses indicated that (103)Pd should be more effective than (125)I because it had less dependence on T(pot). The greatest benefit of (103)Pd was shown to be with tumors with a short T(pot). Although the regrowth delay would be longer with (125)I, the benefit was inconsequential compared with the very slow doubling times of localized prostate cancer. Treatment planning with either isotope revealed no significant differences. These findings may explain why clinically there seemed to be no clear difference in treatment outcome with either isotope. Based on these predictions, we recommend a clinical trial to compare the efficacy of the two isotopes.

Adenocarcinoma↗

PubMed1997

Effects of 42 degrees C hyperthermia on intracellular pH in ovarian carcinoma cells during acute or chronic exposure to low extracellular pH.

PURPOSE: To determine whether intracellular pH (pHi) is affected during hyperthermia in substrate-attached cells and whether acute extracellular acidification potentiates the cytotoxicity of hyperthermia via an effect on pHi. METHODS AND MATERIALS: The pHi was determined in cells attached to extracellular matrix proteins loaded with the fluorescent indicator dye BCECF at 37 degrees C and during 42 degrees C hyperthermia at an extracellular pH (pHe) of 6.7 or 7.3 in cells. Effects on pHi during hyperthermia are compared to effects on clonogenic survival after hyperthermia at pHe 7.3 and 6.7 of cells grown at pHe 7.3, or of cells grown and monitored at pHe 6.7. RESULTS: The results show that pHi values are affected by substrate attachments. Cells attached to extracellular matrix proteins had better signal stability, low dye leakage and evidence of homeostatic regulation of pHi during heating. The net decrease in pHi in cells grown and assayed at pHe = 7.3 during 42 degrees C hyperthermia was 0.28 units and the decrease in low pH adapted cells heated at pHe = 6.7 was 0.14 units. Acute acidification from pHe = 7.3 to pHe = 6.7 at 37 degrees C caused an initial reduction of 0.5-0.8 unit in pHi, but a partial recovery followed during the next 60-90 min. Concurrent 42 degrees C hyperthermia caused the same initial reduction in pHi in acutely acidified cells, but inhibited the partial recovery that occurred during the next 60-90 min at 37 degrees C. After 4 h at 37 degrees C, the net change in pHi in acutely acidified cells was 0.30 pH unit, but at 42 degrees C is 0.63 pH units. The net change in pHi correlated inversely with clonogenic survival. CONCLUSIONS: Hyperthermia causes a pHi reduction in cells which was smaller in magnitude by 50% in low pH adapted cells. Hyperthermia inhibited the partial recovery from acute acidification that was observed at 37 degrees C in substrate attached cells, in parallel with a lower subsequent clonogenic survival.

Animals↗

PubMed1997

Altered proton extrusion in cells adapted to growth at low extracellular pH.

Intracellular pH (pHi) homeostasis is crucial to cell survival. Cells that are chronically exposed to a low pH environment must adapt their hydrogen ion extrusion mechanisms to maintain their pHi in the physiologic range. An important component of the adaptation to growth at low pH is the upregulation of pHi relative to the extracellular pH (pHe). To test the ability of low pHe adapted cells to respond to a pHi lowering challenge, a fluorescence assay was used that directly monitors proton removal as the rate of change of pHi during recovery from cytosolic acidification. Two cell lines of Chinese hamster origin (ovarian carcinoma and ovary fibroblastoid cells) were compared, both of which showed altered proton extrusion after adaptation to growth at low pHe = 6.70. In the ovarian carcinoma (OvCa) cell line, the pattern was consistent with an upregulation by means of an increase in the number of functional proton transporters in the plasma membrane. In the ovary fibroblastoid (CHO-10B) cell line, pHi was consistently elevated in adapted cells as compared with cells grown at normal pHe = 7.30 without an increase in maximum extrusion rate. This upregulation was consistent with a shift in the activating pHi of proton transporters without an increase in the number of transporters, i.e., a change in substrate affinity of the transporter. In OvCa cells, recovery from acidification could be blocked by amiloride, an inhibitor of Na+/ H+ exchange. In contrast, a more modest effect of amiloride on CHO cells was observed but a complete inhibition was seen with the Cl-/HCO(-3)exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). These data indicate that the two cell lines rely to different degrees on the two major pathways for pH regulation during recovery from cytosolic acidification.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1996

Thermotolerance and intracellular pH in two Chinese hamster cell lines adapted to growth at low pH.

As an in vitro model for the low extracellular pH (pHe) which has frequently been observed in tumors, cell lines have been grown in a low-pH medium in order to allow cell adaptation to that milieu. Two Chinese hamster cell lines [Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa)] were compared, both of which acquired thermotolerance during 42 degrees C heating in pHe = 7.3 buffer, but not in pHe = 6.7 medium unless grown at that pH long enough to become adapted. CHO cells, even when acutely acidified, showed higher intracellular pH (pHi) values in a suspension assay than OvCa cells, which confirmed the danger of comparing absolute values of pHi between cell lines. Despite this fundamental difference, relative changes in pHi were similar in that both lines showed a higher pHi in adapted than in unadapted cells, over the range of pHe values tested. The upregulation of pHi was statistically significant, but the two lines differed in the time frame over which adaptation occurred. OvCa cells acquired an enhanced ability to develop tolerance to 42 degrees heat at pHe = 6.7 in 4 days, but the CHO cells acquired this ability more progressively, achieving a maximum ability at approximately 100 days. In contrast, both lines were able to upregulate their pHi within 4 hours of being exposed to pH 6.7 medium. A further indication of different biochemical mechanisms at work was the opposite effects seen on pHi in the two cell lines upon the removal of extracellular CO2/HCO3-. The differential between adapted and unadapted OvCa cells was enhanced by removal of bicarbonate, whereas CHO cells seemed less stable and the data with greater scatter failed to show any difference between adapted and unadapted cells.

Adaptation, Physiological↗

PubMed1995

Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.

Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.

Animals↗

PubMed1995

Thermal response and hyperthermic radiosensitization of scid mouse bone marrow CFU-C.

PURPOSE: Scid mice are severely immunodeficient as a result of a defective recombinase system. Mice with the scid mutation have been shown to have an increased sensitivity to ionizing radiation, presumably as a result of an inability to repair DNA damage. Little is known of the impact of this mutation on the thermal response and on hyperthermic radiosensitization. This investigation established the thermal response (42-44 degrees C), patterns of thermotolerance development, and the impact of hyperthermia (60 min at 40 degrees C or 42 degrees C) on the radiation response of bone marrow colony forming unit-culture cells (CFU-C) in scid mice. METHODS AND MATERIALS: Anesthetized scid mice (pentobarbital, 90 mg/kg) were killed by cervical dislocation and the nucleated marrow obtained from both tibia and femora by passing 2 ml of cold McCoy's 5A medium supplemented with 15% fetal bovine serum through each bone. Single cell suspensions of nucleated marrow were heated in 12 x 75 mm sterile tissue culture tubes at a concentration of approximately 5 x 10(6) cells/ml. Radiation, when used, was delivered immediately prior to hyperthermia by a 137Cs irradiator (dose rate of 1.20 Gy/min). Colony forming unit-culture were cultured in semisolid agar in the presence of colony stimulating factor (conditioned medium from L929 cells) for 7 days. RESULTS: The slope of the radiation dose-response curve for CFU-C in scid mice was biphasic, the Dos (+/- SE) were 0.29 +/- 0.03 Gy and 1.09 +/- 0.20 Gy, respectively. The Dos of the radiation dose-response curve for wild type marrow from CB-17 and Balb/c mice were 1.28 +/- 0.05 Gy and 1.47 +/- 0.15 Gy, respectively. The Dos of the hyperthermia dose-response curves for scid mice were 75 +/- 5, 10 +/- 1.4, and 4 +/- 0.2 min, respectively, for temperatures of 42 degrees, 43 degrees, and 44 degrees C. Thermotolerance development at 37 degrees C increased to a maximum at approximately 240 min after acute hyperthermia (15 min at 44 degrees C) and thereafter, decreased to control levels within 15 h. Thermotolerance did not develop in scid CFU-C during chronic hyperthermia at temperatures < 42.5 degrees C. Hyperthermia (60 min at 40 degrees or 42 degrees C) immediately after ionizing radiation did not significantly alter the terminal slope of the radiation dose-response curve of scid CFU-C (Do = 1.28 +/- 0.08 Gy). By contrast, hyperthermia following radiation of wild type CFU-C resulted in a decrease in the Do from 1.47 +/- 0.05 Gy (Balb/c, rad only) to 1.31 +/- 0.08 or 1.06 +/- 0.18 Gy for 60 min at 40 degrees or 42 degrees C, respectively. CONCLUSION: These results show that the thermal response and the pattern of thermotolerance development of scid CFU-C were similar to that of wild type Balb/c CFU-C, but that hyperthermia given immediately after ionizing radiation did not alter the radiation response of scid CFU-C. The scid mutation does not increase hyperthermic sensitivity or change the pattern of thermotolerance development of scid mouse CFU-C, implying that the scid mutation is not involved with thermal response, but does render the already radiation-sensitive scid cells incapable of thermal radiosensitization.

Animals↗

PubMed1995

Thermoradiotherapy in the management of superficial malignant tumors.

In recent years there have been numerous randomized and nonrandomized studies conducted to assess the efficacy of hyperthermia combined with either radiation therapy or chemotherapy, especially in the treatment of superficially seated malignant tumors. The major impact of hyperthermia is currently on locoregional control of tumor. Heat may be directly cytotoxic to tumor cells or inhibit repair of both sublethal and potentially lethal damage after radiation. These effects are augmented by the physiological conditions in tumors which lead to states of acidosis and hypoxia. Blood flow is often impaired in tumor relative to normal tissue, and hyperthermia may lead to a further decrease in blood flow and augment heat sensitivity. Three major areas of clinical investigation have borne the greatest fruit for hyperthermia as adjunctive therapy to radiation therapy. These include recurrent and primary breast lesions, melanoma, and head and neck neoplasms. The thermal enhancement ratio was increased in all cases and is estimated to be 1.4 for neck nodes, 1.5 for breast, and 2 for malignant melanoma. In general, the most important prognostic factors for complete response are radiation dose, tumor size, and minimal thermal parameters (minimum thermal dose, mean minimum temperature or temperature exceeded by 90% of thermal sensors). The number of heat fractions administered per week appears to have no bearing on the overall response, which may be indicative of the effects of thermotolerance. The total number of heat fractions delivered also appears to be irrelevant provided adequate heat is delivered in one or two sessions. The major prognostic factors for the duration of local control are tumor histology, concurrent radiation therapy dose, tumor depth, and mean minimum temperature.

Animals↗

PubMed1994

Tumor extracellular pH as a prognostic factor in thermoradiotherapy.

PURPOSE: Tumor extracellular pH measurements in 26 human tumors were evaluated for the purpose of prognostic indication of response to thermoradiotherapy. METHODS AND MATERIALS: Twenty-six patients (10 male, 16 female; mean age 62 years, range 18-89) were treated with external microwave hyperthermia (915 MHz) combined with radiation therapy. Tumor histologies included: 46% adenocarcinoma, 38% squamous cell carcinoma, 12% soft tissue sarcoma, and 4% malignant melanoma. The mean tumor depth was 1.6 +/- 0.2 cm (range 0.4-3 cm) and the mean tumor volume was 73 +/- 11 cm3 (range 1-192 cm3). The mean radiation dose administered concurrently with hyperthermia was 39 +/- 1 Gy (range 24-60 Gy, median of 40 Gy), in 15 fractions (range 8-25), over 32 elapsed days (range 15-43). The mean number of hyperthermia sessions administered was 5.4 +/- 0.5 (range 2-10). A battery operated pH meter and combination 21 ga recessed glass, beveled needle microelectrodes were used for tumor pH measurements. Calibration in pH buffers was performed before and after each pH measurement. The needle microelectrodes were 2.5 cm in length. RESULTS: A complete response (CR) was obtained in 20 of 26 patients (77%) and a partial response in six (23%). The mean extracellular tumor pH was 6.88 +/- 0.09 in CR patients while it was 7.24 +/- 0.09 in noncompletely responding (NCR) patients (p = 0.08). Logistic regression analysis indicated that the probability of obtaining a complete response was influenced by the tumor volume (p = 0.02), tumor depth (p = 0.05), and extracellular tumor pH (p = 0.08). Lesions in the pH range of 6.00-6.40 and lesions in the pH range of 6.41-6.80 exhibited a CR rate of 100%, while those lesions in the pH range of 6.81-7.20 exhibited a CR of 90% and those in the pH range of 7.21-7.52 exhibited a CR of 50% (p = 0.002). In lesions with depth < or = 1.5 cm, the CR rate was 100% when the tumor pH was < 7.15 and 75% when the tumor pH was > or = 7.15. In lesions with depth between 1.5 and 3 cm, the CR rate was 66% when the tumor pH was < 7.15 and 43% when the tumor pH was > or = 7.15 (p = 0.02). In small tumors, that is, < or = 20 cm3, tumor pH increased with volume, whereas in larger tumors, that is, > 20 cm3, tumor pH decreased as a function of tumor volume. CONCLUSION: Tumor extracellular pH may be useful as a prognostic indicator of tumor response to thermoradiotherapy.

Combined Modality Therapy↗

PubMed1994

Human tumor extracellular pH as a function of blood glucose concentration.

PURPOSE: Mammalian cells are sensitized to hyperthermia when the extracellular pH (pHe) is acutely reduced to < pH 7.0-7.2. However, cells chronically adapted to low pHe may not demonstrate such sensitivity. Although much of the extracellular environment of human tumors is at lower than normal physiological pH, it may be necessary to acutely acidify tumors to cause a change in the therapeutic response to hyperthermia. The purpose of this study was to reduce extracellular pH in human tumors by elevation of blood glucose. METHODS AND MATERIALS: The change in tumor pHe was measured as a function of the change in blood glucose concentration after oral administration of 100 g glucose in 25 fasting, nondiabetic patients. pHe was determined by needle microelectrodes, and blood glucose determined by "Chemstrips" and a glucometer. In some patients blood glucose concentration rose with time after ingestion to a peak change of 50-100 mg/dL between 30-70 min and then began to decrease. In another group of patients glucose concentration increased by 100-200 mg/dL over 30-90 min and remained elevated as if the patients in this group were Type II diabetics. RESULTS: In 14 transient hyperglycemic patients (56%), as blood glucose increased tumor pHe decreased by a mean of -0.17 +/- 0.04 pH units (p < or = 0.0001, range of -0.41-(+)0.07). By contrast in eight persistent hyperglycemic patients, tumor pHe remained unchanged or actually increased an average of 0.03 +/- 0.04 pH units (range of -0.15-(-)0.14). Normal tissue pHe in five patients was unchanged by hyperglycemia, pHe = 7.33 +/- 0.03. Among all patients, 52% exhibited a pHe decrease > or = 0.1 pH unit, and 24% exhibited a pHe decrease > or = 0.2 pH unit. In five transient hyperglycemic patients whose preglucose tumor pHe was between 6.90 and 7.22, the average decrease in pHe induced by hyperglycemia was 0.25 +/- 0.05 pH unit. A linear relationship was observed between the change of pHe and the maximum change in blood glucose such that the greatest decrease in tumor pHe occurred when the glucose change was minimal. The slope was 0.0017 +/- 0.0005 pH units/mg/dL glucose (p < or = 0.005). The linear relationship included both tumors in transient hyperglycemic patients and in persistent hyperglycemia patients. CONCLUSION: Since patients who exhibited the lowest change in blood glucose exhibited the greatest decrease in tumor pHe, it may be that cells in these patients were better able to transport glucose intracellularly which in tumor cells would permit a more rapid production of lactic acid from aerobic and/or anaerobic glycolysis. These data may be helpful in predicting the response of individual patients to oral hyperglycemia as a clinical thermosensitizer.

Blood Glucose↗