Comparison between the clonogenic, MTT, and SRB assays for determining radiosensitivity in a panel of human bladder cancer cell lines and a ureteral cell line.

Using a series of human bladder cancer cell lines and an immortalised normal ureteral cell line, radiosensitivities measured by three different methods after a single dose of X-radiation are compared. Clear differences between cell survival curves obtained using the clonogenic, microtetrazoline (MTT) and sulforhodamine B (SRB) assays are shown. The most sensitive of the assays investigated was the clonogenic assay. The MTT and SRB assays were found to be relatively insensitive especially at lower radiation levels, suggesting that these assays may not be suitable for predicting therapeutic dose schedules in vivo, but will be important for investigating radio-sensitivity in cell lines with very low plating efficiencies. Each assay discriminated between a range of sensitivities in the cell lines examined, and with some minor differences, the ordering of sensitivities using the three assays was similar. Possible explanations for the differences between results obtained with the three assays are discussed.

Heterogeneity of in vitro radiosensitivity in human bladder cancer cells.

Human bladder cancer is often heterogeneous containing biologically different populations. Radiotherapy plus chemotherapy is the most common treatment for invasive disease. However few studies have investigated the role of heterogeneity in determining radiosensitivity. The radiation sensitivities of a parent human bladder cancer cell line (UCRU-BL-17CL) and nine cloned cell lines derived from it were determined. These cloned cell lines were previously shown to exhibit different biological characteristics when grown in nude mice. Radiation sensitivity was determined using both MTT and clonogenic assays. The radiobiological parameters, alpha,beta, and surviving fractions at 2 Gy and 8 Gy from the linear-quadratic model, were used to assess radiation sensitivity in the statistical analyses. The nine clones differed in radiosensitivity by both assays. By MTT, but not by the clonogenic assay, their radiation sensitivities were relatively consistent within each of the three biological groups (non-tumorigenic, tumorigenic, invasive); invasive clones were more sensitive than those of the non-tumorigenic and the tumorigenic groups for all the three-test criteria. The heterogeneity exhibited by this cell line may explain some of the variations in the clinical responses seen in the radiation treatment of invasive bladder cancer.

A 610 kb YAC clone harbors 7 cM of tomato (Lycopersicon esculentum) DNA that includes the male sterile 14 gene and a hotspot for recombination.

Pollen development requires both sporophytic and gametophytic gene expression. We are using a map-based cloning technique to isolate sporophytic genes which, when mutant, cause pollen abortion and a male sterile (ms) phenotype in tomato (Lycopersicon esculentum). We have genetically characterized one ms locus (ms14) using RFLP analysis and identified flanking markers. High-resolution genomic physical mapping indicates that the ms14 locus is located in a approximately 300 kb region. We have identified a YAC clone with an insert size of approximately 610 kb that contains the ms14-linked markers, reflects the organization of the physical map and therefore most probably contains the ms14 gene. In addition, we present evidence that the relationship between physical and genetic distance in this chromosomal region changes abruptly from approximately 105-140 kb/cM to less than 24kb/cM, and suggest that the TG393-TG104 region is a hotspot for recombination.

Effect of capsaicin on distribution of binding sites for tachykinins and calcitonin gene-related peptide in rat urinary bladder: a quantitative autoradiographic study.

The autoradiographic localization of binding sites for [125I]BH-[Sar9,Met(O2)11]SP, [125I]NKA, and [125I]CGRP was investigated in adjacent sections of urinary bladder body, from adult rats pretreated 14 days before with capsaicin or vehicle. Location of silver grains was assessed both qualitatively and quantitatively using computerized densitometry. Dense labeling of smooth muscle was seen with both [125I]BH-[Sar9,Met(O2)11]SP ([125I]BHSar-SP) and [125I]NKA; in addition, [125I]BHSar-SP labeled submucosal blood vessels. For these radioligands, no differences were apparent between sections from capsaicin- and vehicle-pretreated rats. Specific binding of [125I]CGRP was observed over the epithelium and weakly over submucosal arterioles, but not over smooth muscle. The density of [125I]CGRP binding sites on the epithelium, but not blood vessels, was increased (p < 0.05) by 22% after chronic capsaicin pretreatment, suggesting receptor upregulation. This study demonstrates that although all three peptides are colocalized in primary afferent sensory fibers in rat urinary bladder, the receptors for these neuropeptides are located on different cell types and may be subject to different neural influences.

Isolation of the MHC genes encoding the tumour-specific class I antigens expressed on a murine fibrosarcoma.

The C3H UV-induced fibrosarcoma, 1591, is highly immunogenic and, therefore, is readily rejected when transplanted into immunocompetent syngeneic recipients. Previous analysis of 1591 with tumour-specific or H-2-reactive monoclonal antibodies revealed that this antigenicity might be due to the expression of two novel class I major histocompatibility complex (MHC) antigens. In this report we describe the molecular cloning and initial characterization of three genes which account for all of the unique serological class I reactivities observed on this tumour. These include two distinct, but highly conserved, H-2L-like genes, and a third gene the product of which bears determinants which are characteristic of both the tumour and of class I products of the H-2k haplotype. Moreover, each of these genes contains a polymorphic restriction enzyme fragment which is detected in the class I sequences of 1591 relative to normal C3H tissue. Since the expression of these polymorphic class I sequences is relevant to the immunogenicity of 1591, the mutational events by which these genes were generated may be significant to the immunobiology of this tumour.

Rocky Mountain spotted fever vaccine in an animal model.

Formalin-killed, purified Rickettsia rickettsii vaccine was evaluated in a guinea pig model of R. rickettsii infection. Vaccinated guinea pigs were partially protected by the vaccine when challenged with virulent, viable rickettsiae. Greater protection was observed when higher doses of vaccine were given and when frequent booster injections were administered. Stimulation of cell-mediated immunity to the vaccine antigens was variable and also appeared to be achieved more reproducibly with booster vaccinations. Serum antibody was elicited by high doses of vaccine and by booster vaccinations. The presence of serum antibody was useful in predicting immunity to challenge with R. rickettsii.