Radiobiological evaluation of the suitability of negative pions in tumor therapy; facts and theories

Summary A successful cancer radiotherapy consists mainly in sparing normal tissues and killing malignant cells. Negative pions seem to have several and unique gain factors, as Fowler and Perkins 1961 proposed. With the biomedical pion channel of the 590 MeV proton-accelerator of the Swiss Institute for Nuclear Research (SIN) we had since several years the opportunity to test some theoretical conceptions in comparison to several preliminary experiments performed with pions of low dose rate (Berkeley, CERN, Nimrod). The dosimetric measurements showed for the momentum of 180 MeV/c and a ratio e^–/ ^– of 0.1 an excellent depth curve with p. ex: a peak/plateau ratio of 2.5. Besides this important gain factor for radiotherapy, negative pions have the unique particularity to act on the tissues in the same treatment with two different types of radiations, in the peak (treatment volume) with high LET radiation (presumably high RBE) and low LET radiation in the plateau. Following systems have been used: Inactivation of single mammalian cells, induction of chromatid aberrations in Chinese hamster cells; small intestine of mouse (early and late effects); early and late effects in mouse foot; induction of anomalies in mouse embryos; induction of cerebral microvascular damages in neonatal rats; proliferation of Ehrlichascites carcinoma cells; induction of different types of mutation in different stages of male germ cells and somatic cells (Drosophila). The RBE-values in the peak region vary between 0.7–3.3, and are different even in the same system with the same end point but at different cell stages and conditions. For the plateau region the RBE-values lie mostly under 1 (compared with 140 kV-photons) and can be identical with 29 MeV-photons. The clinically important peak/plateau relation lies in every experiment over 1 and reaches even the value of 4.2. The unexpected RBE-values in peak under 1 lead to a new conception of RBE, the two system theory. In intrinsic radiosensitive euoxic systems (healthy tissue) the RBE of peak (star) pions can be under 1, in intrinsic radioresistant hypoxic systems (tumor cells) in contrary over 1. The two systems can also have different vulnerability of repair svstem.Invited paper, presented at the 14th Annual Meeting of European Society of Radiation Biology, Jülich, Germany, October 8–14, 1978Supported by the Swiss National Science Foundation (grant no. 3.682-0.75)Prof. Dr. A. Prader dedicated on the occasion of his 60th birthday     

Construction of new stable strain over-expressing the glucose isomerase of the Streptomyces sp. SK strain

In order to over express the xylA gene of Streptomyces sp. SK strain, it was cloned under the control of the constitutive ermE-up promoter. This construct was integrated through site-specific recombination process into the chromosome of a Streptomyces violaceoniger glucose isomerase deficient strain using the non-replicative vector pTS55. The resulting CBS4 strain shows a perfect stability in the absence of selection pressure. Its glucose isomerase activity was about four and nine-fold greater, than that obtained from Streptomyces sp. SK, respectively fully induced or not by xylose.     

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes

Summary We have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed.     

Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy

Metastasis is an impediment to the development of effective cancer therapies. Our understanding of metastasis is limited by our inability to follow this process in vivo. Fluorescence microscopy offers the potential to follow cells at high resolution in living animals. Semiconductor nanocrystals, quantum dots (QDs), offer considerable advantages over organic fluorophores for this purpose. We used QDs and emission spectrum scanning multiphoton microscopy to develop a means to study extravasation in vivo. Although QD labeling shows no deleterious effects on cultured cells, concern over their potential toxicity in vivo has caused resistance toward their application to such studies. To test if effects of QD labeling emerge in vivo, tumor cells labeled with QDs were intravenously injected into mice and followed as they extravasated into lung tissue. The behavior of QD-labeled tumor cells in vivo was indistinguishable from that of unlabeled cells. QDs and spectral imaging allowed the simultaneous identification of five different populations of cells using multiphoton laser excitation. Besides establishing the safety of QDs for in vivo studies, our approach permits the study of multicellular interactions in vivo.     

Incorporating fluorescent dyes and quantum dots into magnetic microbeads for immunoassays

Microbeads that are both paramagnetic and fluorescently labeled are commercially available in colors spanning the visible spectrum. Although these commercial beads can be bright, polydispersity in both size and fluorescent intensity limit their use in quantitative assays. Very recently, more monodisperse beads have become available, but their large size and surface properties make them less than ideal for some bioassay applications. Here we describe methods to customize commercial nonfluorescent magnetic microparticles with fluorescent dyes and quantum dots (QDs) without affecting their magnetic or surface chemical properties. Fluorescent dyes and 3.3-nm diameter CdSe/ZnS QDs were sequestered within 0.8-micron diameter magnetic beads by swelling the polystyrene matrix of the bead in organic solvent, letting the chromophores partition, and then collapsing the matrix in polar solvents. Chromophore incorporation has been characterized using both UV-visible absorption spectroscopy and fluorescence microscopy, with an average of 3 x 10(8) rhodamine 6G molecules/bead and 6 x 10(4) QDs/bead. The modified beads are uniform in size and intensity, with optical properties comparable to currently available commercial beads. Immunoassay results obtained with our custom fluorescent magnetic microbeads are consistent with those obtained using conventional magnetic microbeads.     

Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function

Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway.     

Polymorphisms in the RANTES gene increase susceptibility to active tuberculosis in Tunisia

RANTES plays a pivotal role in attracting and activating various leukocyte populations that control Mycobacterium tuberculosis infection. The present study investigated the relationship between the RANTES polymorphisms (-28C/G; rs2280788, and -403G/A; rs2107538) and susceptibility to active tuberculosis (TB) in Tunisian populations. A total of 168 patients with pulmonary TB (pTB), 55 with extrapulmonary TB (epTB), and 150 control subjects were studied. Genotype analyses were carried out using polymerase chain reaction-restriction fragment length polymorphism method. We found that the -28 GG genotype was significantly associated with susceptibility to pTB (odds ratio [OR]=11.19; 95% confidence intervals [CI], 5.14-25; P corrected for the number of genotypes [Pc]=10(-8)) and epTB (OR=11.67; 95% CI, 4.74-29.33; Pc=10(-8)). However, the -28 CC genotype was found to be significantly associated with resistance to pTB (OR=0.08; 95% CI, 0.04-0.16; Pc=10(-8)) and epTB development (OR=0.11; 95% CI, 0.05-0.27; Pc=10(-8)). -403A allele was associated with increased risk development of epTB (OR=2.21; 95% CI, 1.18-4.14; p=0.007). G-G and A-C haplotypes and the AG/GC diplotype were associated with increase susceptibility to pTB (OR=7.88, 95% CI, 5.38-11.55; Pc=3.10(-8); OR=2.32, 95% CI, 1.32-4.11; Pc=3.10(-3); OR=13.26, 95% CI, 6.06-29.89; Pc=3.10(-8); respectively) and epTB (OR=6.64, 95% CI, 4-11.05; Pc=3.10(-8); OR=2.6, 95% CI, 1.26-5.35; Pc=12.10(-3); OR=11.26, 95% CI, 4.44-29.28; Pc=3.10(-8); respectively). Collectively, our findings suggested an association of the RANTES -28C/G and -403G/A functional polymorphisms with susceptibility to Mycobacterium tuberculosis infection in Tunisian populations.