Mode of cell death induced in Chinese hamster cells by sequence-selective DNA minor groove binding nitrogen mustards: comparison with untargeted mustards and with Hoechst 33342

The clinical antitumor efficacy of nitrogen mustards such as chlorambucil may relate to their ability to cause programmed cell death (apoptosis), probably through their DNA cross-linking properties. In contrast, bisbenzimidazoles such as Hoechst 33342 interact noncovalently with the minor groove of DNA, and appear to cause apoptosis in a fundamentally different way, which may involve the inhibition of topoisomerase (topo) I enzymes. A series of DNA minor groove binding nitrogen mustards with selective DNA affinity and in vivo antitumor activity in animal models was studied. Although two examples of such compounds proved to inhibit topo I enzymes in vitro, they were equally toxic towards topo I-proficient and- deficient strains of yeast, suggesting that topo I inhibition was not involved in cell killing. Flow cytometric analysis of Chinese hamster cells highlighted the differences in the propensity to cause apoptosis by chlorambucil compared with Hoechst 33342, revealing two distinct apoptotic populations in cells treated with the latter drug. Unexpectedly, the bisbenzimidazole mustards showed a novel peak of apoptotic activity, distinct from that shown by either parent drug. Exploring these different mechanisms of apoptosis may provide new directions for the development of antitumor drugs.     

P53(110-124)-specific human CD4+ T-helper cells enhance in vitro generation and antitumor function of tumor-reactive CD8+ T cells

Current evidence suggests that the optimal vaccines for cancer should incorporate tumor-specific cytotoxic as well as helper epitopes. Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines, which could combine multiple tumor epitopes defined by CD8(+) CTLs, as well as CD4(+) T-helper cells. To test this possibility, we generated anti-p53 CD4(+) T cells from peripheral blood obtained from an HLA-DRB1*0401(+) donor by in vitro stimulation with dendritic cells and recombinant human p53 protein. We identified the wt p53(110-124) peptide as a naturally presented epitope. In a series of ex vivo experiments, performed in an autologous human system, we then demonstrated the ability of anti-wt p53(110-124) CD4(+) T cells to enhance the generation and antitumor functions of CD8(+) effector cells. The results demonstrate the crucial role of T helper-defined epitopes in shaping the immune response to multiepitope cancer vaccines targeting p53. This model of tumor-specific CD8(+) and CD4(+) T-cell interactions suggests that future vaccination strategies targeting tumor cells should incorporate helper and cytotoxic T cell-defined epitopes.     

Workshop on cancer biometrics: identifying biomarkers and surrogates of cancer in patients: a meeting held at the Masur Auditorium, National Institutes of Health

The current excitement about molecular targeted therapies has driven much of the recent dialog in cancer diagnosis and treatment. Particularly in the biologic therapy of cancer, identifiable antigenic T-cell targets restricted by MHC molecules and the related novel stress molecules such as MICA/B and Letal allow a degree of precision previously unknown in cancer therapy. We have previously held workshops on immunologic monitoring and angiogenesis monitoring. This workshop was designed to discuss the state of the art in identification of biomarkers and surrogates of tumor in patients with cancer, with particular emphasis on assays within the blood and tumor. We distinguish this from immunologic monitoring in the sense that it is primarily a measure of the tumor burden as opposed to the immune response to it. Recommendations for intensive investigation and targeted funding to enable such strategies were developed in seven areas: genomic analysis; detection of molecular markers in peripheral blood and lymph node by tumor capture and RT-PCR; serum, plasma, and tumor proteomics; immune polymorphisms; high content screening using flow and imaging cytometry; immunohistochemistry and tissue microarrays; and assessment of immune infiltrate and necrosis in tumors. Concrete recommendations for current application and enabling further development in cancer biometrics are summarized. This will allow a more informed, rapid, and accurate assessment of novel cancer therapies.     

Modulation of TcR/CD3-zeta chain expression by a circulating factor derived from ovarian cancer patients

In women with ovarian cancer, suppression of components of the immune system may promote tumour development. Previous studies in ovarian cancer have demonstrated that decreased expression and function of the T-cell receptor (TcR)-associated signal transducing zeta-chain correlates with deficient immune responsiveness of T cells. In this study, sera and ascitic fluids obtained from woman with advanced ovarian cancer were found to suppress the expression of TcR-associated zeta chain. This suppression of zeta chain expression was dose-dependent and was not observed with biologic fluids obtained from healthy women. The factor responsible for the loss of zeta chain was purified from ascites and characterized as a protein with an appropriate molecular weight of 14 kD. Suppression of T-cell TcR-zeta was specific, since neither lck nor ZAP-70 expression was affected, while zeta chain was almost completely suppressed. This selective suppression of TcR-zeta expression by the 14 kD ascites-derived factor was shown to operate at the mRNA level. By defining the mechanism through which this protein modulates TcR-zeta chain levels, it might be possible to ultimately prevent the suppressive influences of the tumour microenvironment and restor immune competence in patients with ovarian carcinoma.     

Basal cell carcinomas: association of allelic variants with a high-risk subgroup of patients with the multiple presentation phenotype

Previous studies have shown that patients who present at first or a later presentation with a cluster of new basal cell carcinoma (BCC) comprise a subgroup, termed multiple presentation phenotype (MPP), that is at increased risk of developing further lesions. In this study, we examined the hypothesis that patients who develop multiple clusters are a high-risk subgroup. We found, in a total group of 926 BCC patients, 32 patients with 2-5 BCC clusters (multiple cluster MPP) and 113 cases with only one cluster (single cluster MPP). Multiple cluster MPP cases had mean of 11.3 BCC compared with 3.7 in single cluster MPP cases during similar follow-up. Ultraviolet (UV) exposure in these groups was similar. We determined whether the multiple cluster MPP was associated with characteristics associated with sensitivity to UV or glutathione S-transferase (GST) GSTT1, GSTM1, cytochrome P450 (CYP) CYP2D6, tumour necrosis factor (TNF)-alpha and vitamin D receptor (VDR) genotypes previously associated with BCC presentational phenotypes. While the frequencies of blue eyes and male gender were greater in multiple cluster than single cluster cases, these differences were not significant. In multiple cluster cases, mean age at first presentation with single tumours occurred earlier and the frequencies of CYP2D6 extensive metabolizer (EM) (94.4%) and GSTT1 null (41.2%) were significantly greater (P = 0.028 and P = 0.004) than in single cluster cases (67.1% and 14.3%, respectively). The odds ratios for the individual associations of CYP2D6 EM and GSTT1 null with the multiple cluster MPP were relatively larger; 15.5 and 7.39, respectively. TNF-alpha and VDR genotypes were not associated with multiple cluster MPP. We propose that the MPP is not the consequence of excessive UV exposure but rather reflects the presence of a distinct BCC subgroup which is defined by combinations of risk genes.     

Immunobiology and immunotherapy of head and neck cancer

The development of head and neck cancer (HNC) is strongly influenced by the host immune system. Immunoselection of tumors resistant to immune attack and the ability of established tumors to disarm or eliminate immune cells favor tumor progression. Recent evidence for local as well as systemic apoptosis of T lymphocytes, the paucity of dendritic cells (DC) at the tumor site, or the presence of signaling defects in T lymphocytes of patients with HNC emphasizes the fact that their antitumor responses are compromised. The clinical and biologic importance of these immune biomarkers is revealed by the finding that they appear to independently predict 5-year survival in patients with oral carcinoma. Whereas the mechanisms responsible for immune dysfunction in HNC are being investigated, new immunotherapeutic strategies, including antitumor vaccines and DC-based interventions, aim at the restoration of tumor-targeted immune responses. These novel biologic therapies, alone or in combination with conventional therapies, might be expected to protect immune cells from dysfunction or death and to enhance their antitumor activity.     

Cyclooxygenase 2 mRNA expression in rat brain after peripheral injection of lipopolysaccharide

Inducible cyclooxygenase 2 (COX 2) converts arachidonic acid to prostaglandins, which are thought to mediate various peripheral lipopolysaccharide (LPS)-induced central effects, including generation of fever and activation of the hypothalamic–pituitary–adrenal axis. To localize prostaglandin production in the brain following peripheral LPS administration, COX 2 mRNA expression was examined by in situ hybridization histochemistry in rats injected intraperitoneally (i.p.) or intravenously (i.v.) with various doses of LPS or saline. Constitutive expression of COX 2 mRNA was found in neurons of cortex, hippocampus, and amygdala, but not in cells of the blood vessels. COX 2 mRNA levels were not altered in saline-injected animals as compared to non-injected controls. In LPS-injected animals, no consistent changes of neuronal COX 2 mRNA expression were observed. COX 2 mRNA expression appeared ex novo at 0.5-h post-injection in cells closely associated with blood vessels, however, ex novo labeling of the number of labeled cells increased to a peak at 2 h and subsided gradually to basal levels by 24 h. Initially, labeling was observed in cells comprising major surface-lying blood vessels and meninges. Later, vascular and perivascular cells associated with smaller penetrating blood vessels were labeled. This pattern of COX 2 mRNA induction is independent of the route and dose of the LPS injection. The induced COX 2 mRNA producing cells are identified as endothelial and leptomeningeal cells. Changes in COX 2 mRNA expression were not observed in circumventricular organs. These results suggest that peripheral LPS induces a rapid increase in COX 2 production throughout the vasculatures of the brain, which could affect the neuronal activity of widespread brain regions by elevating the levels of prostaglandins.     

Determination of glomerular permselectivity to neutral dextrans in the dog

The multiple-indicator-dilution (MID) technique was used to separate solute flux (Js) across the glomerular and postglomerular capillaries in vivo. Anesthetized mongrel dogs (n = 20) during mannitol diuresis received a pulse of extracellular indicators including 125I-albumin (plasma reference), [14C]inulin (glomerular reference), creatinine (interstitial reference), and a neutral [3H]dextran (specific mol wt between 10,000 and 24,000 dalton) in the left renal artery. Left renal venous and ureteric outflows were rapidly sampled. 3H-labeled dextrans 10,000-15,500 had renal vein mean transit times (t) greater than those of 125I-albumin, indicating postglomerular extraction. 3H-labeled dextrans greater than 15,500 had t values identical to those of 125I-albumin, indicating only unidirectional glomerular extraction. The glomerular fractional dextran extractions relative to simultaneously injected [14C]inulin (ED/Ei) were calculated from urine and renal vein outflow curves and ranged from 0.98 +/- 0.02 to 0.33 +/- 0.12 (SD) for mol wt 10,000 +/- 24,000. ED/Ei values were quantitatively identical to constant-infusion fractional clearances of the same dextrans. Renal plasma flow (RPF) was then deliberately reduced two-to threefold in the same dog. ED/Ei as measured by the MID technique remained unchanged during graded reduction in RPF. In constant-infusion experiments RPF was reduced from 5.78 to 2.77 ml X s-1 X 100 g-1 and GFR from 50.4 to 36.3 ml X min-1, but the fractional neutral dextran clearances remained constant. The predominance of convective solute flux across the dog glomerulus permitted calculation of glomerular reflection coefficients 0.03 +/- 0.06 to 0.85 +/- 0.03 (SD) for neutral 3H-labeled dextrans 10,000-24,000 dalton.