Targeting proapoptotic protein BAD inhibits survival and self-renewal of cancer stem cells.

Emerging evidence suggests that the resistance of cancer stem cells (CSC) to many conventional therapies is one of the major limiting factors of cancer therapy efficacy. Identification of mechanisms responsible for survival and self-renewal of CSC will help design new therapeutic strategies that target and eliminate both differentiated cancer cells and CSC. Here we demonstrated the potential role of proapoptotic protein BAD in the biology of CSC in melanoma, prostate and breast cancers. We enriched CD44(+)/CD24(-) cells (CSC) by tumorosphere formation and purified this population by FACS. Both spheres and CSC exhibited increased potential for proliferation, migration, invasion, sphere formation, anchorage-independent growth, as well as upregulation of several stem cell-associated markers. We showed that the phosphorylation of BAD is essential for the survival of CSC. Conversely, ectopic expression of a phosphorylation-deficient mutant BAD induced apoptosis in CSC. This effect was enhanced by treatment with a BH3-mimetic, ABT-737. Both pharmacological agents that inhibit survival kinases and growth factors that are involved in drug resistance delivered their respective cytotoxic and protective effects by modulating the BAD phosphorylation in CSC. Furthermore, the frequency and self-renewal capacity of CSC was significantly reduced by knocking down the BAD expression. Consistent with our in vitro results, significant phosphorylation of BAD was found in CD44(+) CSC of 83% breast tumor specimens. In addition, we also identified a positive correlation between BAD expression and disease stage in prostate cancer, suggesting a role of BAD in tumor advancement. Our studies unveil the role of BAD in the survival and self-renewal of CSC and propose BAD not only as an attractive target for cancer therapy but also as a marker of tumor progression.

Saudi Guidelines on the Diagnosis and Treatment of Pulmonary Hypertension: Challenges in managing pulmonary hypertension in the developing countries.

There is scant published data about pulmonary hypertension (PH) from the developing countries. True prevalence of the disease, its biology, etiology and response to treatment are not well known, and they are likely to be somewhat different from that of the developed countries. In this review, we will discuss the main challenges for managing PH in developing countries and propose real-life recommendations to deal with such difficulties.

Comparative evaluation of protective antigen produced from Bacillus anthracis & Escherichia coli.

BACKGROUND & OBJECTIVE: Anthrax has been reported from almost every country and India is endemic for this disease. There is considerable under reporting of the disease because of lack of microbiological facilities and diagnostic reagents. In India only conventional methods which have limitations, are being used to diagnose the disease. Hence the aim of this study was to isolate and purify protective antigen (PA) using different protocols and to use this PA for detection of anti-PA antibodies from sera samples. METHODS: Protective antigen was isolated and purified from the Sterne strain of Bacillus anthracis. B. anthracis lacking pXO1 and pXO2 transformed with pYS5 (B. anthracis pYS5) and recombinant Escherichia coli transformed with pQE30 containing PA gene using hydroxyapatite (HA), Q-sepharose fast protein liquid chromatography (FPLC) and nickel-nitrilotriacetic acid (Ni-NTA) chromatographic methods, respectively. A mixture of PA and edema factor (EF) was injected subcutaneously into rabbits to test the biological activity of PA. The immunogenicity of PA was tested by inoculating the protein into rabbits along with adjuvant. Using this PA, 20 bovine sera samples (pre- and post-vaccinated) were tested by Western blotting (WB) for the presence of anti-PA antibodies. RESULTS: The 83 kDa PA protein was obtained from all the bacteria with the yields of 13, 50 and 9.0 mg/l from Sterne B. anthracis, B. anthracis pYS5 and recombinant Esch. coli, respectively. Formation of edematous ulcers at the site of PA+EF injection clearly confirmed the retention of biological activity of the proteins. Of the 10 post-vaccination sera tested, 9 showed clear positive by WB whereas none of the pre-vaccination sera showed the reaction. INTERPRETATION & CONCLUSION: The purified PA preparations obtained in the present study may possibly be utilized for detection of anti-PA antibodies in the sera of anthrax patients for timely diagnosis of the disease and, might also be tested for their efficacy and use as human anthrax vaccine.

Identification of Bacillus anthracis by a simple protective antigen-specific mAb dot-ELISA.

A simple protective antigen (PA)-reactive mAb dot-ELISA was standardized for confirmation of toxin-producing strains of Bacillus anthracis. Twenty-seven clinical isolates were collected from patients clinically suspected of having anthrax. PA was elaborated from these isolates using Casamino acids medium and the culture medium was boiled to kill the cells. PA in boiled culture supernatants was detected using a dot-ELISA. Of the 27 clinical isolates tested, PA was detected in 24 isolates. This was further confirmed by amplifying the PA gene by PCR. This testing procedure is simple to perform, specific and safer than existing procedures, which are added advantages over existing methods of identification of B. anthracis. This test system could be a valuable tool in confirming clinical and environmental isolates of B. anthracis.

Generation and characterization of monoclonal antibodies to protective antigen of Bacillus anthracis.

Monoclonal antibodies (MoAbs) were generated following immunization of BALB/c mice with protective antigen (PA) of B. anthracis. Five clones reactive to this protein were stabilized and preserved. These MoAbs could detect nanogram levels of PA when tested in ELISA. In Western blotting, they reacted with all PA preparations tested and no cross-reactivity was observed with lethal factor, edema factor of B. anthracis and with other organisms. These MoAbs could detect PA from 22 confirmed clinical isolates of B. anthracis on Western blotting and hold promise for direct detection of PA in clinical samples for diagnosing anthrax.

Relative biological effectiveness of 99mTc radiopharmaceuticals.

The radiotoxicity of three 99mTc-labeled compounds is investigated using spermatogenesis in mouse testis as the experimental model, and spermatogonial cell survival as the biological end point. The radiopharmaceuticals studied are pertechnetate (99mTcO4-), pyrophosphate (99mTc-PYP), and hydroxyethylene diphosphate (99mTc-HDP). The mean lethal doses at 37% survival (D37) are 0.70 +/- 0.06, 0.84 +/- 0.13, and 0.59 +/- 0.08 Gy for 99mTcO4-, 99mTc-PYP, and 99mTc-HDP, respectively. When these results are compared with the D37 value obtained with external x rays or internal gamma rays, the relative biological effectiveness (RBE) of these compounds are 0.94 +/- 0.09, 0.79 +/- 0.13, and 1.1 +/- 0.16, respectively. These results show that the radiotoxicity of 99mTc in mouse testis is essentially similar to that of low-LET radiations (i.e., RBE approximately 1). To understand these results, the distribution of these radiocompounds in the testis is determined and correlated with the observed RBE values. The expected range of RBE values for 99mTc radiopharmaceuticals in organs is 0.95 to 1.5, depending on the fraction of organ activity that is bound to DNA. This suggests that the Auger electrons emitted in the decay of 99mTc are not capable of causing extreme toxicity in vivo. These results provide further support for 99mTc as the radionuclide of choice for imaging in nuclear medicine.

Chromium toxicity in Neurospora crassa.

A comparative study has been made on the mechanisms of toxicities of trivalent and hexavalent forms of chromium in Neurospora crassa. Of the two forms, Cr6+ is more toxic than Cr3+. The toxicity of Cr3+ was found to be due to its specific antagonism with iron uptake. Fe3+ was found to be very effective in reversing the toxicity of Cr3+ by concomitantly suppressing its uptake. That the Cr3+ toxicity caused a conditional intracellular iron deficiency was indicated by the decrease in the activities of catalase and uricase and a progressive increase in the excretion of iron binding compound into the medium. The toxicity of Cr6+ (as Cr2O7(2-)) was found to be due to its specific antagonism of sulfate uptake. Methionine was found to be more effective in reversing dichromate toxicity than sulfate, probably by repressing the synthesis of sulfate permeases responsible for dichromate (Cr6+) uptake. Maximal uptake of Cr6+ was nearly tenfold lower and Vmax much higher than that of Cr3+. Evidence has been adduced to show that Cr6+ and Cr3+ were toxic by themselves and that interconversion between the tri- and hexavalent forms of chromium did not occur to any detectable extent.

Biological effect of lead-212 localized in the nucleus of mammalian cells: role of recoil energy in the radiotoxicity of internal alpha-particle emitters.

The radiochemical dipyrrolidinedithiocarbamato-212Pb(II) [212Pb(PDC)2] is synthesized and its effects on colony formation in cultured Chinese hamster V79 cells are investigated. The cellular uptake, biological retention, subcellular distribution and cytotoxicity of the radiocompound are determined. The 212Pb is taken up quickly by the cells, reaching saturation levels in 1.25 h. When the cells are washed, the intracellular activity is retained with a biological half-life of 11.6 h. Gamma-ray spectroscopy indicates that the 212Pb daughters (212Bi, 212Po and 208Tl) are in secular equilibrium within the cell. About 72% of the cellular activity localizes in the cell nucleus, of which 35% is bound specifically to nuclear DNA. The mean cellular uptake required to achieve 37% survival is 0.35 mBq of 212Pb per cell, which delivers a dose of 1.0 Gy to the cell nucleus when the recoil energy of 212Bi and 212Po decays is ignored and 1.7 Gy when recoil is included. The corresponding RBE values compared to acute external 137Cs gamma rays at 37% survival are 4.0 and 2.3, respectively. The chemical Pb(PDC)2 is not chemotoxic at the concentrations used in this study. Because the beta-particle emitter 212Pb decays to the alpha-particle-emitting daughters 212Bi and 212Po, these studies provide information on the biological effects of alpha-particle decays that occur in the cell nucleus. Our earlier studies with cells of the same cell line using 210Po (emits 5.3 MeV alpha particle) localized predominantly in the cytoplasm resulted in an RBE of 6. These earlier results for 210Po, along with the present results for 212Pb, suggest that the recoil energy associated with the 212Bi and 212Po daughter nuclei plays little or no role in imparting biological damage to critical targets in the cell nucleus.

Radiotoxicity of platinum-195m-labeled trans-platinum (II) in mammalian cells.

The chemotoxicity and radiotoxicity of trans-dichlorodiammineplatinum (II) labeled with 195mPt (trans-195mPt) are investigated to ascertain the potential of radioplatinum coordination complexes as antineoplastic agents. Platinum-195m, with a half-life of about 4 days, is a prolific emitter of low-energy Auger electrons because of the high probability of internal conversion in its isomeric transitions. The kinetics of cellular uptake and retention after incubation and the radiotoxicity of this Auger electron emitter in the form of trans-195mPt is investigated using cells of the Chinese hamster V79 cell line. The cellular uptake of 195mPt reaches a plateau in about 3 to 5 h of incubation and varies nonlinearly with the extracellular concentration of radioactivity. The radioactivity is eliminated from the cells after incubation with an effective half-life of 24 h. Cell survival data, when corrected for the chemical toxicity of nonradiolabeled trans-platinum, give a cell survival curve typical for radiations with high linear energy transfer. At 37% survival, the mean lethal cellular uptake is about 1.0 mBq/cell. Dosimetric considerations, based on subcellular distribution of the radionuclide, yield a value of 4.8 for the relative biological effectiveness when compared with 250 kVp X rays. Theoretical Monte Carlo track-structure calculations indicate that the density of radical species produced in liquid water in the immediate vicinity of a 195mPt decay site is substantially greater than the density of species along the track of a 5.3 MeV alpha particle. This explains qualitatively the efficacy of 195mPt in causing high-LET radiation type biological effects. The extreme radiotoxicity of intranuclearly localized 195mPt, in conjunction with the proclivity of platinum chemotherapy agents to bind to DNA in the cell nucleus, suggests that the combination of chemical effects and the effects of Auger electrons that can be obtained with radioplatinum coordination complexes may have potential in the treatment of cancer.

Vitamins as radioprotectors in vivo. I. Protection by vitamin C against internal radionuclides in mouse testes: implications to the mechanism of damage caused by the Auger effect.

The potential of vitamin C, an antioxidant, to protect the radiosensitive spermatogonial cells in mouse testes against the effects of chronic irradiation by radionuclides incorporated into tissue was investigated. Interestingly, when injected intratesticularly, a small and nontoxic amount of vitamin C (1.5 microgram in 3 microliters saline) protected the spermatogonia against the damage associated with high-LET radiation caused by Auger electrons from similarly administered 5-(125I)-iodo-2'-deoxyuridine (125IdU). A dose modification factor (DMF) of 2.3 was obtained. In contrast, no protection was observed when 210Po, an alpha-particle emitter, was administered similarly. These findings suggest that the mechanism of action of the Auger effect is of an indirect nature, which is in contrast to the direct action generally believed to be responsible for biological damage caused by high-LET radiations.

Vitamin C as a radioprotector against iodine-131 in vivo.

The capacity of vitamin C (ascorbic acid) to mitigate radiation damage resulting from the tissue-incorporated radionuclide 131I is examined. Spermatogenesis in mice is the experimental model and spermhead survival is the biological endpoint. When a small nontoxic amount of vitamin C was injected, followed by a similar injection of 131I, the 37% spermhead survival dose (D37) increased by a factor of 2.2 compared with the D37 in animals receiving only the radionuclide. Similar radioprotection was also observed when the animals were maintained on a diet enriched with 1% vitamin C (by weight). These results suggest that vitamin C may play an important role as a radioprotector against accidental or medical radiation exposures, especially when radionuclides are incorporated in the body and deliver the dose in a chronic fashion.

On the equivalent dose for Auger electron emitters.

Radionuclides that emit Auger electrons are widely used in nuclear medicine (e.g., 99mTc, 123I, 201Tl) and biomedical research (e.g., 51Cr, 125I), and they are present in the environment (e.g., 40K, 55Fe). Depending on the subcellular distribution of the radionuclide, the biological effects caused by tissue-incorporated Auger emitters can be as severe as those from high-LET alpha particles. However, the recently adopted recommendations of the International Commission on Radiological Protection (ICRP) provide no guidance with regard to calculating the equivalent dose for these radionuclides. The present work, using spermatogenesis in mouse testis as the experimental model, shows that the lethality of the prolific Auger emitter 125I is linearly dependent on the fraction of the radioactivity in the organ that is bound to DNA. This suggests that the equivalent dose for Auger emitters may have a similar linear dependence. Accordingly, a formalism for calculating the equivalent dose for Auger emitters is advanced within the ICRP framework.

Radiotoxicity of some iodine-123, iodine-125 and iodine-131-labeled compounds in mouse testes: implications for radiopharmaceutical design.

In this work, spermhead survival in mouse testis was used to investigate the radiotoxicity of several intratesticularly localized radioiodinated pharmaceuticals. Radioiodines that decay by electron capture and/or internal conversion (123I, 125I) as well as by beta- decay (131I) were coupled to pharmaceuticals that selectively localize in different cell compartments. Dose response curves yield D37 values of 62 cGy, 75 cGy, 61 cGy and 7.7 cGy for 123IMP (N-isopropyl-p-iodoamphetamine), 131IdU (iododeoxyuridine), H131IPDM (N,N,N'-trimethyl-N'-(2-hydroxyl-3-methyl-5-iodobenzyl)-1,3-propanediami ne) and 125IdC (iododeoxycytidine), respectively. At 37% survival, the relative biological effectiveness (RBE) of these radiochemicals, when compared to the pure gamma-emitting radiochemical 7Be-chloride (D37 = 65 cGy), are 1.0, 0.89, 1.1 and 8.4, respectively. Intratesticular 7Be, with an effective half-life of 430 hr in the organ, was used as the source of reference radiation to determine the RBE values because it solely emits 477 keV gamma rays, and the dose to the testis is delivered chronically, as in the case of the other radiocompounds. Subcellular distribution studies show that all of the cellular activity is localized in the cytoplasm in the cases of 123IMP and H131IPDM, while virtually all of 131IdU and 125IdC were bound to DNA in the cell nucleus. In agreement with our earlier in vivo studies, these data show that subcellular distribution plays a key role in the radiotoxicity of Auger electron emitters such as 123I and 125I, and has no role for beta emitters such as 131I. These findings may have implications in the design of radiopharmaceuticals for both diagnosis (localize Auger emitter in cytoplasm of cell) and therapy (localize Auger emitter in cell nucleus).

Biological effects of the Auger emitter iodine-125: a review. Report No. 1 of AAPM Nuclear Medicine Task Group No. 6.

The biological implications of Auger electron cascades following inner shell ionization of atoms have been of interest for over 25 years. By virtue of their decay via orbital electron capture and/or internal conversion, several biomedical radionuclides emit numerous low-energy electrons spontaneously. The biological effects of such radionuclides incorporated into tissues cannot be predicted a priori because of the highly localized patterns of energy deposition by the electrons. Results of extensive research using Iodine-125 as a model Auger electron emitter are now available. This article presents an up-to-date review of the physical and radiobiological data on this Auger emitter. Valuable concepts concerning the action of internal Auger emitters are identified phenomenologically, and questions that need to be answered are indicated. The present understanding provides a scientific basis toward estimation of risk associated with Auger emitters used in diagnosis, and suggests potential applications to therapy.

The question of relative biological effectiveness and quality factor for auger emitters incorporated into proliferating mammalian cells.

The problem of determining RBE values for Auger emitters incorporated into proliferating mammalian cells is examined. In general, the reference radiation plays a key role in obtaining experimental RBE values. Using survival of cultured Chinese hamster V79 cells as the experimental model, new data are provided regarding selection of a reference radiation for internal Auger emitters. These data show that gamma rays delivered acutely (137Cs) are more than twice as lethal as gamma rays delivered chronically with an exponentially decreasing dose rate (99mTc). The results confirm that the reference radiation should be delivered chronically in a manner consistent with the extended exposure received by the cells in the case of incorporated radionuclides. Through a direct comparison of the radiotoxicity of Auger emitters and alpha emitters, the high RBE values reported for DNA-bound Auger emitters are confirmed. These studies reveal that the DNA binding compound [125I]iododeoxyuridine (125IdU) is about 1.6 times more effective in killing V79 cells than 5.3 MeV alpha particles from intracellularly localized 210Po-citrate. In addition, toxicity studies with the radiochemicals 125IdU and [125]-iododeoxycytidine (125IdC) establish the equivalence of the radiosensitivity of thymine and cytosine base sites in the DNA. In view of these results, and information already available, the question of establishing quality factors for Auger emitters is considered. Finally, a method for calculation of the dose equivalent for internal Auger emitters is advanced.

Induction of sperm head abnormalities by incorporated radionuclides: dependence on subcellular distribution, type of radiation, dose rate, and presence of radioprotectors.

In contrast to the biological effects caused by exposure to external beams of radiation, the effects of tissue-incorporated radionuclides are highly dependent on the type of radiation emitted and on their distribution at the macroscopic, microscopic, and subcellular levels, which are in turn determined by the chemical nature of the radionuclides administered. Induction of abnormalities of sperm heads in mice is investigated in this work after the injection of a variety of radiochemicals including alpha emitters. When the initial slopes of the dose-response curves are used to compare the relative biological effectiveness (RBE) of different radiocompounds, the alpha particles emitted in the decay of 210Po are more effective than Auger electrons emitted by 125I incorporated in the DNA of the spermatogonial cells, and both emissions are more effective than X rays. It is also shown that the Auger emitters (125I, 111In) distributed in the cell nucleus are more efficient in producing abnormalities than the same radionuclides localized in the cytoplasm. These findings are consistent with our earlier observations, where spermatogonial cell survival is assayed as a function of the testicular absorbed dose. Further, chronic irradiation of testis with gamma rays from intratesticularly administered 7Be is about three times more effective in causing abnormalities than a single acute exposure to 120-kVp X rays. The resulting RBE values correlate well with our data on sperm head survival with the same radiocompounds. Finally, the radioprotector cysteamine, when administered in small, nontoxic amounts, significantly reduces the incidence of sperm abnormalities from alpha-particle radiation as well as emissions from 125I incorporated into DNA, the dose reduction factors being 10 and 14, respectively.

Biological consequence of nuclear versus cytoplasmic decays of 125I: cysteamine as a radioprotector against Auger cascades in vivo.

When the radionuclide 125I is localized in mouse testis as 125I-iododeoxyuridine (an analogue of thymidine) and incorporated into the DNA of spermatogonial cells, the cytocidal effects are as severe as those due to densely ionizing alpha particles. In contrast, 125I confined to the cytoplasm of these cells is much less radiotoxic, the efficacy being the same as for selective irradiation of the testis with sparsely ionizing external X rays. The biological effects, in both cases, are strongly mitigated upon pretreatment of the testes with very small amounts (0.75 microgram) of cysteamine, a radioprotector. These findings suggest an important role for such chemical agents in radiation protection and in understanding the mechanisms of radiation damage involving radionuclides incorporated in tissue.

In-vivo radiotoxicity of DNA-incorporated 125I compared with that of densely ionising alpha-particles.

When the atomic nucleus of 125I decays by orbital electron capture followed by internal conversion, numerous very-low-energy electrons (Auger electrons) are emitted, so that the energy density in the immediate vicinity of the decay site is extremely high. 125I incorporated into DNA was as effective as densely ionising 5.3 MeV alpha-particles from 210Po in reducing the sperm-head population in mice. Hence the biological risks of Auger-electron emitting radionuclides widely used in biology and medicine ought to be reassessed.

Radiotoxicity of 5-[123I]iodo-2'-deoxyuridine in V79 cells: a comparison with 5-[125I]iodo-2'-deoxyuridine.

The toxic effects of the short-lived (T 1/2 = 13.2 h) Auger-electron-emitting isotope 123I, incorporated in the form of 123IUdR into the DNA of V79 cells in vitro, have been investigated and compared to those of 125IUdR. For the concentrations tested, the rate of incorporation of 123IUdR at any time is proportional to the concentration of extracellular radioactivity. The curve for survival of clonogenic cells decreases exponentially and exhibits no shoulder at low doses. The mean lethal dose (D37) to the nucleus is 79 +/- 9 cGy and is about the same as that obtained previously with 125IUdR. However, the total number of decays needed to produce this D37 with 123IUdR is about twice that required with 125IUdR, approximately equal to the ratio of the energy deposited in microscopic volumes by 125I and 123I, respectively. This correlation suggests that nuclear recoil, electronic excitation, and chemical transmutation are probably of minor importance to the observed biological toxicity with either isotope. The results also indicate that there are no saturation effects in the decay of 125IUdR in the DNA of V79 cells (i.e., all of the emitted energy is biologically effective) and that each of the two steps involved in the 125I decay is equally effective in causing biological damage.