A diamond target for megavoltage cone-beam CT.

PURPOSE: To determine the properties of a megavoltage cone-beam CT system using the unflattened beam from a sintered diamond target at 4 and 6 MV. METHODS: A sintered diamond target was used in place of a graphite target as part of an imaging beam line (an unflattened beam from a graphite target) installed on a linear accelerator. The diamond target, with a greater density than the graphite target, permitted imaging at the lower beam energy (4 MV) required with the graphite target and the higher beam energy (6 MV) conventionally used with the tungsten/stainless steel target and stainless steel flattening filter. Images of phantoms and patients were acquired using the different beam lines and compared. The beam spectra and dose distributions were determined using Monte Carlo simulation. RESULTS: The diamond target allowed use of the same beam energy as for treatment, simplifying commissioning and quality assurance. Images acquired with the diamond target at 4 MV were similar to those obtained with the graphite target at 4 MV. The slight reduction in low energy photons due to the higher-Z sintering material in the diamond target had minimal effect on image quality. Images acquired at 6 MV with the diamond target showed a small decrease in contrast-to-noise ratio, resulting from a decrease in the fraction of photons in the beam in the energy range to which the detector is most sensitive. CONCLUSIONS: The diamond target provides images of a similar quality to the graphite target. Diamond allows use of the higher beam energy conventionally used for treatment, provides a higher dose rate for the same beam current, and potentially simplifies installation and maintenance of the beam line.

The use of hormonal therapy with radiotherapy for prostate cancer: analysis of prospective randomised trials.

In 1901, Wilhelm Conrad Röntgen won the Nobel prize in Physics for his discovery of the Röntgen rays or, as he himself called them, X-rays. In 1966, Dr Charles Brenton Higgins won the Nobel Prize in Medicine for his breakthroughs concerning hormonal treatment of prostatic cancer. After 31 years, in 1997, the first prospective randomised trials of the combination of hormonal therapy and radiation therapy were published, showing increased survival when compared to radiation therapy alone for patients with prostate cancer. Since 1997, many investigators have published trials combining hormonal and radiation therapy for prostate cancer. This minireview will address the largest and most influential of these trials, and attempt to guide physicians in selecting the appropriate patients for this combined approach.

Evaluation of ultrasound-based prostate localization for image-guided radiotherapy.

To evaluate the use of the ultrasound-based BAT system for daily prostate alignment. Prostate alignments using the BAT system were compared with alignments using radiographic images of implanted radiopaque markers. The latter alignments were used as a reference. The difference between the BAT and marker alignments represents the displacements that would remain if the alignments were done using ultrasonography. The inter-user variability of the contour alignment process was assessed. On the basis of the marker alignments, the initial displacement of the prostate in the AP, superoinferior, and lateral direction was -0.9 +/- 3.9, 0.1 +/- 3.9, and 0.2 +/- 3.4 mm respectively. The directed differences between the BAT and marker alignments in the respective directions were 0.2 +/- 3.7, 2.7 +/- 3.9, and 1.6 +/- 3.1 mm. The occurrence of displacements >/=5 mm was reduced by a factor of two in the AP direction after the BAT system was used. Among eight users, the average range of couch shifts due to contour alignment variability was 7, 7, and 5 mm in the antero-posterior (AP), superoinferior, and lateral direction, respectively. In our study, the BAT alignments were systematically different from the marker alignments in the superoinferior, and lateral directions. The remaining random variability of the prostate position after the ultrasound-based alignment was similar to the initial variability. However, the occurrence of displacements >/=5 mm was reduced in the AP direction. The inter-user variation of the contour alignment process was significant.

p27Kip1 is required for PTEN-induced G1 growth arrest.

The tumor suppressor PTEN is one of the most commonly inactivated genes in human cancer. Glioblastoma multiforme cells harboring mutant PTEN have abnormally high levels of 3' phosphoinositides and elevated protein kinase B activity. Expression of wild-type PTEN in glioma cells, containing endogenous mutant PTEN, reduces 3' phosphoinositides levels, inhibits PKB activity, and induces G1 cell cycle arrest. We investigated the mechanism of the PTEN-induced growth arrest in glioma cell lines. Expression of PTEN is associated with increased expression of p27Kip1, decreased expression of cyclins A and D3, inhibition of cdk2 activity, and dephosphorylation of pRb. Inactivation of p53, by the human papilloma virus E6 oncoprotein, does not prevent PTEN-induced G1 arrest, implying that p53 is not required for G1 arrest. In contrast, p27Kip1 antisense oligonucleotides abrogated the growth arrest induced by PTEN. Furthermore, blocking p27Kip1 expression prevented the PTEN-induced reduction of cyclin-dependent kinase 2 activity, indicating that p27Kip1 functions upstream of cyclin-dependent kinase 2 in the PTEN regulatory cascade. These results implicate p27Kip1 as a critical mediator of PTEN-induced G1 arrest.

Loss of PTEN facilitates HIF-1-mediated gene expression.

In glioblastoma-derived cell lines, PTEN does not significantly alter apoptotic sensitivity or cause complete inhibition of DNA synthesis. However, in these cell lines PTEN regulates hypoxia- and IGF-1-induced angiogenic gene expression by regulating Akt activation of HIF-1 activity. Restoration of wild-type PTEN to glioblastoma cell lines lacking functional PTEN ablates hypoxia and IGF-1 induction of HIF-1-regulated genes. In addition, Akt activation leads to HIF-1alpha stabilization, whereas PTEN attenuates hypoxia-mediated HIF-1alpha stabilization. We propose that loss of PTEN during malignant progression contributes to tumor expansion through the deregulation of Akt activity and HIF-1-regulated gene expression.

Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl-XL.

Prolonged (>24 h) exposure to anti-IgM (an antigen surrogate that induces membrane cross-linking and apoptosis) induced a 3-fold increase in the mass of endogenous ceramide measured by 32P labeling by diacylglycerol kinase and a 4-fold increase in ceramide as measured by metabolic labeling with [3H]palmitate in a B-lymphocyte cell line, WEHI 231. This correlated with the induction of apoptosis. Shorter exposure times to anti-IgM (up to 8 h) failed to elicit apoptosis and did not elicit increased ceramide formation. After 8 h, apoptosis occurs concomitantly with ceramide formation over the next 40 h. Further, we showed that exogenous ceramide mimicked anti-IgM-induced apoptosis and that apoptosis was potentiated in serum-free media. Treatment of cells with an inhibitor of ceramide catabolism, N-oleoylethanolamine, increased both ceramide formation and apoptosis and accelerated apoptosis induced by anti-IgM. To examine further how ceramide metabolism is involved in apoptosis, we derived cell lines from a small population of cells resistant to N-oleoylethanolamine. These cell lines were selected based on an altered ceramide metabolic pathway, were resistant to apoptosis induced by anti-IgM, and showed no significant increase in ceramide when challenged with anti-IgM. The basis of this resistance was shown to be the failure to activate neutral sphingomyelinase activity following 24-h treatment with anti-IgM, in contrast to the 2-fold increase in neutral sphingomyelinase activity observed in wild type cells. We have shown previously that transfection of WEHI cells with bcl-xL conferred resistance to anti-IgM-induced apoptosis, whereas transfection with bcl-2 did not (Gottschalk, A., Boise, L., Thompson, C., and Quintans, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7350-7354). In this study, these bcl-xL transfectants also displayed increased resistance to exogenous N-acetylsphingosine (C2-ceramide) or N-hexanoylsphingosine (C6-ceramide). However, when challenged with anti-IgM the bcl-xL transfectants produced levels of ceramide similar to wild type cells, suggesting that ceramide formation is upstream of bcl-xL and that it is a major determinant of B-cell death.

The ability of Bcl-x(L) and Bcl-2 to prevent apoptosis can be differentially regulated.

Although expression of Bcl-2 has been shown to prevent apoptosis under many circumstances, there are several systems in which Bcl-2 fails to promote cell survival. We have previously reported that Bcl-2 and Bcl-x(L) display differential ability to protect WEHI-231 cells from multiple inducers of apoptosis. A possible explanation for this paradox was provided by the discovery of Bax. Bax is a Bcl-2-related protein which can inhibit the ability of Bcl-2 to enhance the survival of growth factor-dependent cell lines in the absence of growth factor. Consistent with the possibility that Bcl-2 function in WEHI-231 cells is inhibited by Bax, WEHI-231 cells were found to express a high level of Bax. To directly test the effects of Bax expression on Bcl-x(L) function, FL5.12 cells were transfected with both genes. Although Bax overexpression can inhibit Bcl-2 from prolonging cell survival upon growth factor withdrawal, Bax overexpression did not inhibit Bcl-x(L) from preventing apoptosis in this cell line. Although Bcl-2 and Bcl-x(L) were both found to be able to form heterodimers with Bax, the majority of Bax in both cases was not complexed to a partner. Our data suggest that Bcl-x(L) does not function by simply preventing the formation of Bax homodimers which promote cell death. Instead Bax appears to display selectivity in its ability to inhibit Bcl-2 but not Bcl-x(L) from prolonging survival. Furthermore, our data suggest that the abilities of Bcl-2 and Bcl-x(L) to promote cell survival are not identical and can be independently regulated within a cell. Regulation of a cell's apoptotic threshold is likely to result from a complex set of interactions among Bcl-2 family members and other, as yet uncharacterised, regulators of apoptosis.

The role of bcl-XL in CD40-mediated rescue from anti-mu-induced apoptosis in WEHI-231 B lymphoma cells.

The phenotypically immature B cell lymphoma WEHI-231 undergoes apoptotic cell death when cultured with anti-immunoglobulin (Ig) antibodies, via a bcl-2-independent mechanism. We have therefore studied the role of the bcl-2-related protein bcl-x in controlling cell death in WEHI-231. We find that overexpression of the long form of bcl-x (bcl-XL) renders these cells refractory to anti-Ig-induced cell death. Stimulation of WEHI-231 via CD40 has similar protective effects. We show here that ligation of CD40 rapidly induces the appearance of the bcl-XL protein in WEHI-231, while stimulation via sIgM, sIgD, CD5 or CD45 receptors, or with phorbol esters plus ionomycin does not. WEHI-231 cells also rapidly undergo massive apoptosis following culture with thapsigargin, a specific inhibitor of the Ca(2+)-ATPase of the endoplasmic reticulum: this is also reversed by anti-CD40, or by overexpression of bcl-XL. We, therefore, conclude that bcl-XL plays a key role in the regulation of antigen receptor-mediated apoptosis via CD40 in WEHI-231. In addition, the fact that this protein is not induced in WEHI-231 in response to phorbol dibutyrate plus ionomycin points to a fundamental signaling defect in these cells, which could conceivably be a reflection of their immature, apoptosis-susceptible phenotype.

Resistance to anti-IgM-induced apoptosis in a WEHI-231 subline is due to insufficient production of ceramide.

We describe the properties of a physiological cell death (PCD)-resistant subline of WEHI-231 generated from the PCD-susceptible WEHI-231.7 JM cell line maintained in our laboratory. The PCD-resistant WEHI-231.7 JMRE subline was uniquely resistant to anti-immunoglobulin (Ig)M-induced PCD but not to irradiation and etoposide. In these sublines, we compared the expression of genes implicated in regulating PCD. Northern analysis of c-myc, c-fos, egr-1, Fas, p53 and retinoblastoma revealed similar basal levels of expression in all sublines tested and comparable responses to anti-IgM treatment. Similarly, the expression of bcl-2, bcl-x, bax and IL-1 beta converting enzyme did not correlate with susceptibility to anti-IgM-induced PCD. Next, we systematically studied signal transduction events including: tyrosine phosphorylation, Ca++ flux, and ceramide production in the Jm and JMRE sublines. The tyrosine phosphorylation patterns and the Ca++ influx generated following sIgM engagement were very similar in the JM and JMRE sublines. In contrast, the generation of ceramide differed in the PCD-resistant and PCD-susceptible sublines. Ceramide is produced following cross-linking sIgM on WEHI-231.7 JM cells and causes PCD. Ceramide levels in anti-IgM-treated WEHI-231.7 JMRE cells are low and appear to be insufficient to induce PCD.

Apoptosis in B lymphocytes: the WEHI-231 perspective.

In this review we summarize recent work on the molecular basis of apoptosis in the murine B cell lymphoma WEHI-231. WEHI-231 cells undergo apoptosis in response to antigen receptor cross-linking with anti-Ig reagents. Death is specifically triggered via surface IgM (sIgM); cross-linking sIgD, Ia or FcR has no effect. Apoptosis is preceded by growth arrest in the G0/G1 phase of the cell cycle and may not occur in all currently available WEHI-231 sublines. The continuous passage of WEHI-231 cells in different laboratories has yielded variants that differ greatly in their response to anti-Ig treatment because apoptotic cells tend to be negatively selected in culture. Resistant and susceptible variants undergo growth arrest in response to anti-Ig but only susceptible cells go on to die by apoptosis. Cells resistant to anti-Ig have intact apoptotic machinery as indicated by their susceptibility to dexamethasone, irradiation and other treatments. However, anti-Ig-resistant cells are also resistant to apoptosis induced by the immunosuppressants cyclosporin A, FK-506 and rapamycin. We discuss the experimental evidence indicating that the apoptotic machinery in WEHI-231 cells is pre-activated but under constant negative regulation by short-lived protein inhibitors. Inhibition is removed by a mediator released in response to anti-Ig treatment in susceptible sublines. The mediator of death is the sphingosine derivative, ceramide, presumably produced by membrane sphingomyelinases activated by anti-Ig. A hypothetical model on how ceramide kills WEHI-231 is presented.

Bcl-2 and Bcl-2-related proteins in apoptosis regulation.

In this review we have discussed the importance of Bcl-2 and related proteins in the regulation of apoptotic cell death in mammalian systems. It is clear that Bcl-2 plays a critical role in controlling many forms of PCD. Bcl-2 seems to have particular significance in lymphocyte development and the function of the immune system. We have also discussed the increasing size of the newly identified Bcl-2 family. There are a number of Bcl-2 homologues in human, murine, avian, nematode, and viral systems. The evolutionary conservation of the function of the Bcl-2 homologues, reinforces the importance of PCD in all complex organisms. Some of these bcl-2-like genes function as agonists and others as antagonists. Despite the seemingly universal importance of Bcl-2, it is unable to prevent PCD in all systems. In addition, we have described a role for other Bcl-2 family members in systems in which Bcl-2 is ineffective and supplied a potential rationale for the large number of genes involved in the regulation of PCD. Identification and functional analysis of the Bcl-2 family members reveals the complex nature of cell death regulation. As we begin to appreciate the significance of PCD in the control of development and homeostasis, its regulation at the molecular level is becoming better understood. Bcl-2 has long been the only known intracellular regulator of the PCD pathway(s), although its ability to prevent apoptosis is not universal. We now know that bcl-2 is only one member of an evolutionary conserved family of genes which display different patterns of expression as well as function. At least two family members, Bcl-xs and Bax, act in opposition to Bcl-2. The discovery of these new family members, including those with Bcl-2-like function and antagonists, should help clear up the discrepancies seen in Bcl-2's ability to protect cells from PCD. In doing so, we will be able to further define the pathways associated with cell death signaling. The study of these family members, as well as the non-related genes of the PCD pathways (ced-3, ced-4, ice) should lead us to understanding of how cells of multicellular organisms make decisions to die.

Studies on the induction of apoptosis in WEHI 231 cells by pharmacological agents and lipid mediators. Sphingosine and ceramide induce apoptosis in WEHI 231.

We report the results of a systematic study of the effects of pharmacological agents known to cause or modify physiological cell death (PCD). Using WEHI 231 cells as a model, we investigated the effects of dexamethasone, cAMP, selected growth factors/ cytokines, DNA damaging agents, metabolic inhibitors and lipid mediators. We found that WEHI 231 cells are not affected by cAMP(1-90 microM) or TGFbeta (1-50 ng/ml), both of which are known to induce PCD in other systems. We also failed to detect protection from PCD in WEHI 231 cells cultured with Zn(++), E64 and leupeptin. In contrast, dexamethasone (400 microg/ml), etoposide (10(-4)M), emetine (10(-5)M), calyculin (10(-5)M), sphingosine (8-16 microM) and ceramide (20-40 microM) all cause PCD in WEHI 231 cells. The effects of ceramide can be blocked by LPS but not by overexpression of bcl2.The role of killer lipids in PCD is discussed.

Ceramide mediates the apoptotic response of WEHI 231 cells to anti-immunoglobulin, corticosteroids and irradiation.

We demonstrate for the first time how immature B cells kill themselves. Ceramide is identified as the mediator of apoptosis in the murine B lymphoma line WEHI 231 commonly used as a model to study clonal deletion in B lymphocytes. We show that exogenous ceramide induces apoptosis in WEHI 231 cells. To maintain self tolerance, immature lymphocytes readily undergo apoptotic death in response to the cross-linking of their antigen-specific receptors. We demonstrate that endogenously produced ceramide accumulates in WEHI 231 cells exposed to anti-IgM, an antigen surrogate before the onset of apoptosis. We also show that two other inducers of apoptosis, irradiation and dexamethasone, cause intracellular accumulation of ceramide.

Identification of immunosuppressant-induced apoptosis in a murine B-cell line and its prevention by bcl-x but not bcl-2.

Cyclosporin A, FK-506, and rapamycin are immunosuppressants often used as pharmacological probes to study lymphocyte activation and physiological cell death (PCD). Because cyclosporin A and FK-506 are known to prevent PCD in T-cell hybridomas and thymocytes, we used these reagents, as well as rapamycin, to determine whether they alter the pathway leading to apoptosis in murine WEHI-231 cells following surface IgM cross-linking. We observed that the immunosuppressants themselves induced PCD in WEHI-231 cells, but only in sublines susceptible to anti-IgM-mediated apoptosis. PCD was preceded by growth arrest and characterized by the DNA fragmentation pattern typical of apoptosis. In B-cell lines resistant to anti-immunoglobulin- and immunosuppressant-induced PCD, cyclosporin A, FK-506, and rapamycin caused growth arrest. PCD was also induced by inhibitors of protein synthesis in WEHI-231 cells but not in the mature B-cell line BAL-17. Immunosuppressant-induced and protein synthesis inhibitor-induced PCD, but not growth arrest, could be prevented by the overexpression of bcl-xL, while transfection with bcl-2 did not affect PCD or cell cycle arrest. These results suggest that bcl-2 and bcl-xL may control partially independent systems to inhibit PCD in lymphoid cells and that PCD in B and T cells may be differentially regulated.

Fc gamma RII cross-linking inhibits anti-Ig-induced erg-1 and erg-2 expression in BCL1.

Activation of B cells through their Ag receptor is known to be negatively regulated by Fc gamma RII engagement. To explore the molecular and biochemical mechanisms of Fc gamma RII-mediated inhibition, we investigated the effect of Fc gamma RII engagement on the expression of two immediate-early genes, egr-1 and egr-2, and tyrosine phosphorylation events following the activation of the murine B cell line, BCL1. Egr-1 and egr-2 were expressed in BCL1 after slg cross-linking. The induction of egr-1 and egr-2 expression was prevented when the Fc gamma RII was co-cross-linked with slg in BCL1, but not in WEHI-231. The inhibitory effects of Fc gamma RII engagement on egr-1 and egr-2 expression occurred when the Fc gamma RII was cross-linked with either slgM or slgD. Treatment with cyclosporin A prevented the expression of egr-2 induced by slg cross-linking, but did not inhibit egr-1 expression. In addition, cyclosporin A did not prevent the negative-regulatory effects of Fc gamma RII engagement on egr-1 expression, suggesting that the Fc gamma RII works upstream from the site of action of cyclosporin A. To investigate activation signals more proximal to the plasma membrane, we compared tyrosine phosphorylation patterns of several effector molecules known to play a role in B cell activation. Cross-linking of slg induced tyrosine phosphorylation of the p62 GAP-associated protein. The p62 protein became hyperphosphorylated in response to co-cross-linking of slg with Fc gamma RII. Our results identify egr-1 and egr-2 as targets of Fc gamma RII-mediated inhibition of anti-Ig-induced B cell activation. In addition, they show that negative regulation by Fc gamma RII is effective in both cyclosporin A-sensitive and insensitive pathways. Further, we suggest a possible Fc gamma RII signaling pathway leading to the inhibition of egr-1 and egr-2 expression.

Physiological cell death in B lymphocytes: I. Differential susceptibility of WEHI-231 sublines to anti-Ig induced physiological cell death and lack of correlation with bcl-2 expression.

WEHI-231 is a murine lymphoma generally considered to represent an immature B cell. Cross-linking of slg on WEHI-231 leads to growth arrest and eventually physiological cell death (PCD). We characterized three sublines of WEHI-231 by flow cytometry and compared their responses with slg cross-linking. All sublines had identical expression of a series of common B cell surface markers (IgM, IgD, Fc gamma R, ICAM-1, and CD45), but one was I-A-. Despite the phenotypic similarities between these sublines, anti-IgM caused aptotosis in only two sublines, although it inhibited growth in all three. The growth arrest induced by anti-IgM was reversible by lipopolysaccharide and Th2 clones and independent of Fc gamma R engagement. Anti-IgD, unlike anti-IgM, induced neither growth arrest nor apoptosis. To further compare the sublines' susceptibility to PCD, we investigated their responses to anti-IgM by ultrastructural morphology, [3H]thymidine release, propidium iodide exclusion, and incorporation into DNA. By all these experimental criteria, two of the WEHI-231 sublines were susceptible to PCD while the third demonstrated remarkable resistance to anti-IgM, but not irradiation or Th1-induced PCD. This differential susceptibility to PCD did not correlate with either bcl-2 levels in the resting cells or to the decrease in bcl-2 expression following slg engagement. We discuss the implications of these findings for our understanding of PCD in B cells.

Differential induction of Egr-1 expression in WEHI-231 sublines does not correlate with apoptosis.

To determine whether the absence of inducible Egr-1 expression correlates with apoptosis and growth arrest, we compared the inducible expression of two Egr family members (Egr-1 and Egr-2) in three sublines of WEHI-231. Expression of Egr-2 is induced in all sublines of WEHI-231 following surface immunoglobulin (sIg) cross-linking, but Egr-1 expression is induced in only two. We find that the lack of inducible Egr-1 expression corresponded to an increase in the methylation pattern of the Egr-1 gene. In spite of these differences in Egr-1 expression, all the sublines demonstrate similar inhibition of [3H] thymidine incorporation following anti-Ig treatment. Growth arrest leads to apoptosis in only two of the sublines, but apoptosis does not correlate with the absence of inducible Egr-1 expression. Demethylation, by treatment with 5-azacytidine, in the Egr-1 non-expressing subline allows for induction of Egr-1 expression by anti-Ig, but fails to prevent growth arrest and apoptosis. Therefore, we conclude that the lack of Egr-1 expression is not responsible for either the apoptotic response or growth arrest induced by anti-Ig in WEHI-231.