First clinical evaluation of the QIAreachTM QuantiFERON-TB for tuberculosis infection and active pulmonary disease.

OBJECTIVE: 1) to compare the QIAreachTM QuantiFERON-TB (QIAreach QFT) vs. QuantiFERON®-TB Gold Plus assay (QFT-Plus) to detect tuberculosis (TB) infection; 2) to evaluate diagnostic sensitivity of QIAreach QFT using active TB as surrogate for TB infection; 3) to preliminarily evaluate QIAreach QFT in immunocompromised individuals. METHODS: QIAreach QFT measures the level of interferon-γ (IFN-γ) in plasma specimens from blood stimulated by ESAT-6 and CFP-10 peptides in one blood collection tube (equivalent to the TB2 tube of the QFT-Plus). QIAreach QFT was applied to plasma samples from 41 patients with pulmonary TB and from 42 healthy or low-TB-risk individuals. RESULTS: Sensitivity and specificity of QIAreach QFT vs. QFT-Plus were 100% (41/41) and 97.6% (41/42), respectively; overall concordance was 98.8% (82/83). All samples were measured within 20 min. The time to result of each sample was significantly correlated with IFN-γ level with a natural logarithmic scale (r = -0.913, p < 0.001). Seven cases in the active TB group were immunocompromised (CD4 <200/µL) and tested positive by QIAreach QFT. CONCLUSIONS: QIAreach QFT provides an objective readout with a minimum blood sample volume (1 mL/subject), potentially being a useful point-of-care screening test for TB infection in high-TB-burden, low-resource countries and for immunocompromised patients.

Strain dependency of B and T lymphoma development in immunoglobulin heavy chain enhancer (E mu)-myc transgenic mice.

The transgenic mice were produced by injecting eggs of B6 and C3H/HeJ mice with the human E mu-myc gene. Preferential development of B lymphomas was observed in the B6 transgenic mice, whereas the C3H/HeJ transgenic mice developed mostly T lymphomas. The phenotypic activation of B lineage cells but not of T lineage cells was detected in the prelymphomatous transgenic mice of both strains. The transgene was similarly expressed in B and T cells of the transgenic mice of both strains. These results suggest that a high incidence of T lymphomas in the C3H/HeJ transgenic mice may not be due to the preferential activation of or the preferential E mu-myc expression in T lymphocytes. When the bone marrow or fetal liver cells from the prelymphomatous transgenic mice of both strains were transferred into irradiated normal C3H/HeJ mice, most of the recipients developed T lymphomas. Moreover, even when irradiated B6 mice received the hematopoietic stem cells from the prelymphomatous B6 transgenic mice, the incidence of T lymphoma increased up to 50%. These findings suggest that B6 and C3H/HeJ mice might provide the environment that supports the development or growth of B and T lymphomas, respectively, and that such an environment could be modified by irradiation of the mice.

The level of expression of adenovirus type 2 transforming genes governs sensitivity to nonspecific immune cytolysis and other phenotypic properties of adenovirus 2-simian virus 40-transformed cell hybrids.

Syrian hamster embryo cells transformed by adenovirus type 2 (Ad2) or simian virus 40 (SV40) differ markedly in morphology, tumorigenicity, and susceptibility to in vitro lysis by nonspecific cytotoxic cells. Hybrid cells formed by fusing Ad2- and SV40-transformed Syrian hamster embryo cells may express only SV40 T antigens or both SV40 and Ad2 T antigens. Hybrids that express only SV40 T antigens are indistinguishable from the nonhybrid SV40-transformed phenotype, whereas hybrid cells that express T antigens from both viruses closely resemble the nonhybrid parental Ad2-transformed phenotype. Because these hybrid cells have been useful in the study of neoplastic transformation, we determined the amount of viral antigens that they accumulate in an attempt to correlate the level of expression of the transforming viral genes with some of their phenotypic properties. Hybrid cells that expressed proteins from both viruses showed reduced levels of SV40 T antigens compared with those of hybrid cells that did not express Ad2 T antigens. We also found that the production of several cellular proteins that influence cytomorphology was inhibited in hybrid and nonhybrid cells that expressed Ad2 T antigens, and the repression of these cellular proteins correlated with a change in cytomorphology from fibroblastic to spherical. Finally, we showed that the susceptibility of our hybrid cells to in vitro lysis by natural killer cells and activated macrophages, two putative host-effector cells involved in defense against neoplasia, correlated closely with the level of expression of a 58,000-dalton Ad2 protein. The results reported here, together with the results of previous studies, indicate that the oncogenic potential of hybrid cells that express both Ad2 and SV40 antigens is extremely sensitive to Ad2 expression, whereas other phenotypic properties depend on Ad2 expression in a dose-dependent manner.

A novel tetracycline-dependent transactivator with E2F4 transcriptional activation domain.

A tetracycline-controlled gene expression system provides a powerful tool to dissect the functions of gene products. However, it often appears difficult to establish cell lines or transgenic animals stably expressing tetracycline-dependent transactivators, possibly as a result of toxicity of the transactivator domains used. In order to overcome this problem, we developed a novel tetracycline-dependent transactivator that works efficiently in mammalian cells. This transactivator is a fusion of the tet reverse repressor mutant and the transcriptional activating domain of human E2F4, which is ubiquitously expressed in vivo. We demonstrate here that this tetracycline-regulated gene expression system provides a two log transcriptional activation in mammalian cells as assessed by northern blot and luciferase analyses. Combining this system with green fluorescent protein reporter systems or microarray gene expression profiling will facilitate the study of gene function.

Purification and properties of acid ribonucleases in human serum and leukocytes.

Acid RNase was purified from normal human serum about 2400-fold by chromatography on phosphocellulose and Sephadex G-75 and rechromatography on Sephadex G-75. Assayed with yeast RNA as substrate, the enzyme showed the maximal activity at about pH 6.5 with sodium phosphate buffer. The reaction was activated by Na+, K+, and spermine, but it was not affected greatly by Mg2+, Co2+, and EDTA. Ca2+, Fe2+, Zn2+, and Cu2+ inhibited the reaction. Among the synthetic substrates examined, the enzyme preferentially hydrolyzed pyrimidine nucleotides, with a higher affinity for polycytidylate than for polyuridylate. The enzyme was thermolabile, but it stabilized with bovine plasma albumin. The molecular weight was approximately 15,000, estimated gel filtration on Sephadex G-75, and its isoelectric pH was above 11.0. From normal human leukocytes, acid RNase was purified about 400-fold by the same procedure described previously except that rechromatography on Sephadex G-75 was omitted. The properties of leukocytic RNase were found to be similar to those of serum acid RNase, but the latter enzyme differed in substrate specificity substantially from leukocytic RNase, preferring polyuridylate to polycytidylate. This evidence shows that serum RNase is not of leukocytic origin under normal physiological conditions.

Mitogenic and antimitogenic transforming growth factors secreted by adenovirus 2- and simian virus 40-transformed hamster cells: possible roles in promoting tumorigenesis.

Adenovirus 2 (Ad2)- and simian virus 40 (SV40)-transformed hamster embryo cells differ markedly in a number of phenotypic properties including their potential for inducing tumors in hamsters. Both Ad2- and SV40-transformed cells are immortalized and readily induce tumors in immunoincompetent newborn syngeneic hamsters, but only SV40-transformed cells are highly oncogenic in both adult syngeneic and allogeneic immunocompetent hamsters. The reasons for the difference in the oncogenic potential of the Ad2- and SV40-transformed phenotypes remain elusive. However, recent studies with transforming growth factors (TGFs) indicate that these factors play an important role in determining many phenotypic characteristics of transformed cells. To determine whether TGFs secreted by Ad2- and SV40-transformed hamster embryo cells differ, we have examined the ability of media conditioned by these two transformed cell phenotypes to modulate thymidine uptake in quiescent, untransformed cells. We found that both transformed phenotypes secrete very similar TGF alpha-like mitogenic factors which inhibit binding of 125I-labeled epidermal growth factor to its receptor. Our results also show that SV40-transformed cells, but not Ad2-transformed cells, secrete a powerful mitogenic inhibitor (MI). The MI secreted by SV40-transformed cells is inhibitory for several transformed and untransformed cell types and exerts a cytostatic, not cytolytic, action on untransformed primary hamster embryo cells. MI elutes from size exclusion high-performance liquid chromatography columns with a molecular weight of 24,000. Although MI has about the same molecular weight as TGF beta, it differs from TGF beta in two important respects: it is heat labile and it has a different target specificity for antimitogenic activity. The MI secreted by SV40-transformed cells also inhibits thymidine uptake by concanavalin A-stimulated spleen lymphocytes. This finding suggests that MI might contribute to the extreme oncogenicity of SV40-transformed cells by inhibiting mobilization of immune effector cells at the site of tumor cell proliferation.

Serum acid ribonuclease in myelogenous leukemia.

Acid and alkaline RNase activities in serum were measured with yeast RNA as the substrate in normal subjects and in leukemic patients pretreatment and posttreatment, and the acid/alkaline ratios of activities were 0.63 +/- 0.08 (S.D.) (N, 12), 2.28 +/- 0.82 (N, 8), and 0.60 +/- 0.13 (N, 9), respectively. The mean value for the ratio in the pretreated leukemia was significantly higher than that in the other 2 groups (p less than 0.01). By separating these acid and alkaline RNases from normal and leukemic sera by phosphocellulose chromatography, it was further confirmed that acid RNase alone increased markedly in leukemic serum. From serum and leukocytes of leukemic patients, acid RNases were purified about 2000-fold and 300-fold, respectively, by phosphocellulose and Sephadex G-75 chromatography. Both enzymes displayed properties nearly identical with those of normal serum and leukocytes, except that leukemic serum acid RNase had about a 2.4-fold greater affinity for polyuridylate than for polycytidylate as substrate, in contrast to normal serum acid RNase that degraded polycytidylate exclusively. On the other hand acid RNases from serum leukocytes of leukemia showed a similar substrate preference. These results suggest that the high RNase levels of leukemic sera are due to an excessive leakage of acid RNase into the blood stream from abnormal leukocytes.

Change in ascorbate radical production in an irradiated experimental tumor with increased tumor size.

We have reported that ascorbate radical (Asc.-) could serve as an indicator of the amount of hydroxyl radical and superoxide produced by irradiation in vivo. Using this method, we investigated the relationship between tumor size and Asc.- production after irradiation (10 Gy) and between tumor size and the radical-scavenging ability of WR-2721 (300 mg/kg). Asc.- was measured in normal muscle and SCC-VII tumors transplanted into mice (n = 6). In tumors, the increase in Asc.- significantly decreased with increasing tumor size (r = -0.483; P < 0.05). The increase in Asc.- production after irradiation was more inhibited by WR-2721 in normal muscle tissue than in tumor tissue at various sizes. In tumors, the increase in Asc.- was less inhibited by WR-2721 with increasing tumor size. These results demonstrate that the increase in radical production after irradiation and drug distribution decreased with increasing tumor size and that WR-2721 has excellent differential protection. This method is expected to measure changes in the amounts of local hydroxyl radical and superoxide modified by a change of tumor environment or drug administration.

MTG8 proto-oncoprotein interacts with the regulatory subunit of type II cyclic AMP-dependent protein kinase in lymphocytes.

AML1-MTG8 chimeric oncogene is generated in acute myelogenous leukemia with t(8;21), and seems to be responsible for the pathogenesis of the disease. However, the role of MTG8 is ambiguous. Here we found that MTG8 interacted with the regulatory subunit of type II cyclic AMP-dependent protein kinase (PKA RIIalpha). The binding site of MTG8 was NHR3 domain, and that of RIIalpha was the N-terminus for interacting with PKA anchoring proteins (AKAPs). NHR3 contains a putative alpha-amphipathic helix which is characteristic in binding of AKAPs with RII. Indirect immunofluorescence microscopy showed that MTG8 and RIIalpha were overlapped at the centrosome-Golgi area in lymphocytes. These findings suggest that MTG8 may function as an AKAP at the centrosome-Golgi area in lymphocytes.

Correlation between Ca(2+) oscillation and cell proliferation via CCK(B)/gastrin receptor.

Gastrin stimulates cell proliferation through the CCK(B) receptor coupled to Gq-protein, whereas the m3 muscarinic receptor, which also couples to Gq, has no trophic effects. In order to elucidate the cause of the difference, we stably transfected CHO cells with human CCK(B) and m3 receptors. Stimulation of the CCK(B), but not the m3 receptor increased cell growth. Activation of MAP kinase via the m3 receptor was to the same extent as that via CCK(B), indicating that there is an initial signaling common to both receptors. Stimulation of either receptor induced a transient increase in [Ca(2+)](i) followed by a sustained plateau phase. After 2 h of stimulation, the [Ca(2+)](i) response to the m3 receptor disappeared, whereas that to the CCK(B) receptor remained as a [Ca(2+)](i) oscillation. Removal of extracellular Ca(2+), which abolished [Ca(2+)](i) oscillation, completely inhibited DNA synthesis via CCK(B). When the C-terminal part of the CCK(B) receptor was truncated, the trophic effect as well as the [Ca(2+)](i) response after 2 h of stimulation disappeared, whereas the chimeric CCK(B) receptor with the C-terminal region of the m3 receptor preserved its ability to elicit both DNA synthesis and [Ca(2+)](i) oscillation. These results suggest that desensitization might be a principal determinant of cell proliferation, and the persistence of the [Ca(2+)](i) response as [Ca(2+)](i) oscillation could be essential for this type of signal transduction.

Purification and properties of alkaline ribonuclease from human serum.

1. Five alkaline ribonucleases (EC 3.1.4.22) were purified about 140- to 1900-fold from human serum by phosphocellulose and DEAE-cellulose chromatographies and Sephadex G-75 filtration, with a total recovery of 22%. These were designated as RNAases 1-5. 2. Optimum activities were observed at pH 8.5-8.7 for RNAases 1-4, and at pH 7.5 for RNAase 5. The molecular weights of these enzymes were estimated by gel filtration as 45 000, 32 000, 20 000, 13 000 and 8500, respectively. 3. These RNAases were found to be heat-labile proteins but are markedly stabilized with bovine plasma albumin. The reaction was activated by Na+, K+, Mg2+ and Ca2+, and inhibited by Co2+, Fe2+, Cu2+ and Zn2+. EDTA had little effect on the velocity of the reaction. Spermine caused 2- to 7-fold activation. 4. Among the substrates examined, these RNAases preferentially hydrolyzed pyrimidine bodies and except for RNAase 5 had a higher affinity for poly(C) than poly(U) as substrate. Each enzyme was free from other nucleolytic enzymes and hydrolyzed only RNA.

Purification and properties of deoxyribonuclease from human urine.

The DNAase in human urine was purified about 30-fold with a recovery of 28%. This involved DEAE-cellulose and phosphocellulose chromatography steps and gel filtration on Sephadex G-75. The enzyme required divalent cations such as Co2+, Mg2+, Mn2+ and Zn2+ for activity, but Ca2+, Cu2+ and Fe2+ were ineffective. EDTA and G-actin inhibited the reaction. The maximum activity was observed at pH 5.5 in acetate buffer plus Co2+ or Mg2+ and Ca2+. It had a molecular weight of approximately 38 000, estimated by gel filtration on Sephadex G-75 and isoelectric point of around pH 3.9. The enzyme is an endonuclease which hydrolyzes native, double-stranded DNA about 3 to 4 times faster than thermally denatured DNA to produce 5'-phosphoryl- and 3'-hydroxyl-terminated oligonucleotides. The final preparation was free of non-specific acid and alkaline phosphatases, phosphodiesterase and ribonuclease activities.

Primary adult T cell leukemia of bone: two patients with primary bone lesion showing monoclonal integration of HTLV-I proviral DNA.

Adult T cell leukemia (ATL), a neoplasm of mature helper T lymphocytes is etiologically associated with human T lymphotropic virus type-I (HTLV-I). ATL cells infiltrate various organs, the lung, skin, central nervous system, gastrointestinal tract, and bone, causing various clinical manifestations. Two unusual cases of ATL, in which lytic bone lesion was the primary site of ATL, are described. One patient had multiple lytic lesions in bones without any involvement of other organs, and the other patient had a bone lesion in the right radius, which disappeared after chemotherapy. In both cases, monoclonal integration of HTLV-I provirus was demonstrated in the genomic DNA from each bone lesion. Although their clinical courses and pathological findings were different, ATL in both patients began as a bone lesion, showing that primary lymphoma of bone can be manifested in ATL cases.

Detection of K-ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: correlation with clinicopathological features.

We investigated the presence of K-ras gene mutation in plasma DNA and assessed its clinical value in patients with pancreatic adenocarcinoma. Mutations in codon 12 of the K-ras gene were examined by mutant allele-specific amplification method using DNA extracted from surgical specimens and plasma samples of 21 patients with pancreatic adenocarcinoma. K-ras gene mutation was detected in 15 of 21 (71%) primary tumors. In 9 of 15 (60%) patients with K-ras gene mutation-positive tumors, an identical mutation was detected in the plasma DNA. None of four patients with chronic pancreatitis or five healthy subjects had such mutations in plasma DNA. Tumors positive for K-ras gene mutation in plasma DNA were significantly larger (P = 0.04) and less likely to result in a curative cure after surgical resection (P = 0.09) than those negative for the mutation. Other clinicopathological features, including age, sex, histological type, mode of invasion, and metastasis, did not correlate with K-ras gene mutations in plasma DNA. Treatment resulted in disappearance of K-ras gene mutations in plasma DNA in six of nine (67%) patients. Three patients with a persistently positive K-ras gene mutation in pre- and post-treatment plasma samples were likely to show early recurrence or have a progressive disease. Our findings suggest that K-ras gene mutation can be detected in plasma DNA of patients with pancreatic adenocarcinoma. Detection of K-ras mutations in plasma may be clinically useful for evaluating tumor burden and efficacy of treatment.

Glucocorticoid-induced increase in plasma corticosteroid-binding globulin levels in fetal sheep is associated with increased biosynthesis and alterations in glycosylation.

In fetal sheep, there is a concomitant prepartum rise in cortisol and corticosteroid-binding globulin (CBG) that maintains a low free plasma cortisol level and allows for a low negative feedback effect of cortisol on the secretion of ACTH from the fetal pituitary. However, the stimulus for the prepartum increase in CBG and the mechanism(s) of this effect are not known. It has been proposed that glucocorticoids increase CBG concentrations, and therefore, we infused fetal sheep with the synthetic glucocorticoid dexamethasone (DEX; 2 micrograms/min over 15 min every 2 h for 96 h, n = 5) or saline (n = 5). The plasma corticosteroid-binding capacity increased from 30.0 +/- 2.4 to 55.6 +/- 7.7 and 92.6 +/- 11.1 ng/ml at 48 and 96 h, respectively, of DEX infusion. To examine possible mechanisms of increasing fetal plasma CBG, we first cloned and sequenced a sheep CBG cDNA and purified the protein. This allowed us to deduce the primary structure of ovine CBG and to demonstrate that hepatic CBG mRNA abundance (single transcript of 1.8 kilobases) rose from 0.9 +/- 0.2 to 3.6 +/- 1.6 arbitrary units after 96 h of DEX treatment. Fetal DEX treatment produced a significant increase (7.1 +/- 1.2% to 13.1 +/- 1.4%) in the Concanavalin-A-binding forms of CBG that predominate in adult sheep plasma. There was negligible transfer of purified [125I]CBG from the ewe to fetal plasma, urine, or amniotic fluid. We also injected adult sheep with DEX (10 mg/day for 4 days) and demonstrated a significant decrease in plasma corticosteroid-binding capacity by 24 h, which remained suppressed for the duration of the study. After 96 h of DEX treatment, there was also a significant decrease in adult hepatic CBG mRNA abundance. We conclude that glucocorticoids increase fetal plasma CBG in part by increased hepatic biosynthesis. It may also be accentuated by a change in the glycosylation of CBG, but cannot be attributed to transplacental transfer. Furthermore, glucocorticoid treatment exerts opposite effects on CBG biosynthesis in fetal and adult sheep.

Gastrin-releasing peptide-like immunoreactivity is present in human maternal and fetal placental membranes.

Extracts of human term amnion, placenta, and chorion/decidual tissue (n = 5) contained gastrin-releasing peptide-like immunoreactivity (GRPLI) in amounts of 4.7 +/- 2.9 (pmol/g wet wt; mean +/- SEM), 3.6 +/- 1.1 and 2.9 +/- 1.5, respectively. Using C-terminally directed antisera and gel filtration chromatography and reverse-phase high-performance liquid chromatography (HPLC), each tissue contained molecular forms consistent with the presence of GRP1-27 and GRP18-27 but also contained larger amounts of two GRPLI peaks, which apparently are novel GRP-like peptides. In contrast, tissue extracts of human fetal lung contained only GRP1-27, GRP14-27, and GRP18-27. Using RT-PCR and specific GRP primers and probes, messenger RNA encoding for GRP was readily demonstrable from 6-weeks gestation throughout pregnancy to term in full-thickness membranes, placental villi, and decidua. Positive immunohistochemical staining for GRP occurred in extravillous trophoblasts in decidual septa and fetal membranes, cytotrophoblasts, syncytiotrophoblast, and certain stromal cells in placental villi and amniotic epithelium. GRPLI and GRP messenger RNA were present from the earliest dates examined (6-9 weeks) throughout pregnancy to term. Given the proven trophic nature of GRP and related peptides, these peptides may play important roles in maternal, placental, and fetal development during human pregnancy.

A new dosing regimen in renal insufficiency: application to cephalexin.

We describe a new method of drug dosage adjustment. The method simultaneously considers glomerular and tubular functions as parameters, because nonparallel decreases in both functions limit the use of the conventional endogenous creatinine clearance (CLCR) method for dosage adjustment. In the new method, CLCR and the 15-minute phenolsulfonphthalein (PSP15') test were used and applied to patients with renal insufficiency with cephalexin (CEX) as a model drug for renal tubular secretion. The results clearly demonstrate good control of plasma CEX concentrations by the CLCR-PSP15' method, whereas there were marked changes in plasma CEX levels with the CLCR method alone. Our method appears to be more useful for patients with renal impairment than the conventional CLCR method for CEX, which is mainly excreted in urine by renal tubular secretion. A nomogram for the CEX dosing interval is proposed for application to clinical practice.

Enhancement of radiation effects by acyclovir.

Acyclovir (ACV), a new antiviral drug, was used to investigate its effect of radiosensitivity in tumors in vivo. In in vivo experiments with Sarcoma-180 transplanted into the ICR mouse and FM3A transplanted into the C3H mouse, ACV enhanced the radiosensitivity of both tumors. In S-180, radiation effects were enhanced by treatment with 100 mg/kg of ACV from 30 min before to 60 min after irradiation. In S-180 treated by 400 mg/kg of ACV, the enhancement ratio was approximately 2.0, as evaluated by the growth delay method. In the FM3A tumor treated by 20 mg/kg of ACV, the enhancement ratio was approximately 1.3, as evaluated by tumor cure (TCD50 assay). ACV is already clinically used as an antiviral drug. Its ability to radiosensitize tumors could therefore have clinical potential when combined with radiotherapy.

Hydralazine at thermoradiotherapy: tumor size and blood flow effects.

PURPOSE: This study was aimed to assess the dependence on tumor size and blood flow of the efficacy of a vasoactive drug hydralazine with thermoradiotherapy. METHODS AND MATERIALS: Experiments were performed on mice bearing SCC-VII tumors with volumes of about 85 and 340 mm3 (7-8 or 11-12 days after transplantation, respectively). Local hyperthermia (water bath, 43 degrees C, 0.5 h) was started 3 h after irradiation of tumors. Hydralazine (2.5 mg/kg, IP) was given 0.5 h before heating. Tumor blood flow was evaluated by laser Doppler flowmetry before, during and up to 2 days after the treatments. RESULTS: It was shown that hydralazine and hyperthermia, even in combination with each other, had very weak anti-tumor effect, especially for 85 mm tumors. The agents also insignificantly enhanced the efficacy of radiotherapy excluding the case of polyradiomodification for 340 mm3 tumors when a dose modifying factor of about 2.0 was achieved. Thermometry showed only a small improvement by HDZ in heating patterns of tumors of both sizes. Meanwhile, the therapeutic efficacy of hydralazine and heat was correlated with the changes in tumor blood flow, first of all with the delayed effects. The radiomodifiers induced only minor and transient suppression of perfusion in the smaller tumors, and more markedly and for longer time decreased blood flow in the larger tumors. In the latter case, the inhibiting effect of the drug plus hyperthermia remained for at least 48 h after the treatment. CONCLUSION: (a) The combined use of hydralazine and heat seems to be advisable only at radiotherapy of rather large advanced tumors; (b) the efficacy of such radiomodification is correlated with prolonged inhibition of tumor blood flow by these agents; and (c) hydralazine and hyperthermia are likely to kill selectively both acutely and chronically hypoxic radioresistant cancer cells.