Thapsigargin analogues for targeting programmed death of androgen-independent prostate cancer cells.

A number of analogues of thapsigargin, a selective inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPases have been synthesized. In all of the prepared analogues the butanoyl residue at O-8 has been replaced with a residue containing an aromatic amine. The amine can be used as an anchoring point for attaching a peptide group sensitive to the proteolytic enzyme, prostate specific antigen, secreted by prostate cancer cells. Like thapsigargin, the analogues are capable of elevating the cytoplasmic Ca2+ concentration approximately sevenfold when tested at effective cytotoxic doses. The analogues in which the 8-O-butanoyl group has been replaced with 3-(4-aminophenyl)propanoyl or 4-aminocinnamoyl were found potently to induce programmed cell death of the prostate cancer cells.

ACTA, a fluorescent analogue of thapsigargin, is a potent inhibitor and a conformational probe of skeletal muscle Ca2+-ATPase.

Thapsigargin is a highly potent and selective inhibitor of sarco-endoplasmic reticulum (SERCA) family of Ca2+-ATPases and a useful tool in research concerning the function of intracellular Ca2+ stores. We describe here a novel fluorescent derivative (8-O-(4-aminocinnamoyl)-8-O-debutanoylthapsigargin, termed ACTA) of this compound, acting as a Ca2+-ATPase inhibitor with a potency approaching that of thapsigargin. Binding of ACTA to the skeletal muscle sarcoplasmic reticulum vesicles results in a strong fluorescence enhancement, approximately 66% of which depends on ACTA association with Ca2+-ATPase. This specific component of ACTA fluorescence is sensitive to the E1-E2 conformational equilibrium of the pump. The combined properties of high potency and binding-dependent fluorescence suggest ACTA to be a useful probe for a range of studies involving the SERCA class of ATPases.

Irradiation protects against pancreatic islet degeneration and hyperglycaemia following streptozotocin treatment of mice.

Five daily injections of streptozotocin (40 mg/kg) produce islet inflammation, necrosis of pancreatic B cells and hyperglycaemia in the mouse. Anti-pancreatic autoimmunity has been suggested as part of the cause of these events. We have studied the possible effect of total-body irradiation in long-term studies (246 days) and report here that insulitis, islet necrosis and insulin depletion are reduced after irradiation. In parallel the level of hyperglycaemia is reduced. It is concluded that immunological mechanisms are to some extent responsible for the development of streptozotocin-induced diabetes.

Absence of H-2 genetic influence on streptozotocin-induced diabetes in mice.

Five daily injections of streptozotocin (40 mg/kg) produced a delayed but progressively increasing level of hyperglycaemia in long term studies with male Naval Medical Research Institute mice and C3D2F1 (DBA 2 J male x C3H/Tif female) F1 hybrid mice. The development of hyperglycaemia was paralleled by decreased amounts of pancreatic immunoreactive insulin as well as degranulation and necrosis of pancreatic B cells. Insulitis was found from days 9-25 after the first injection of streptozotocin. Compared with the F1 hybrid strain the parental inbred strains DBA 2 J and C3H/Tif demonstrated a certain resistance to streptozotocin. Development of hyperglycaemia did not differ in four congenic resistant lines of mice on the C57 BL/10 genetic background, indicating the major histocompatibility complex genes are not likely to determine susceptibility to streptozotocin-induced islet B cell damage.

The low dose streptozotocin murine model of type 1 (insulin-dependent) diabetes mellitus: studies in vivo and in vitro of the modulating effect of sex hormones.

The influence of sex on pancreatic islet B cell susceptibility to streptozotocin was studied in mice given multiple low doses of streptozotocin. Male C3 D2 F1 mice developed a steadily increasing blood glucose level after a lag period of about 3 weeks, in contrast to females who were resistant. Spleen cells from streptozotocin treated female animals produced hyperglycaemia in total body irradiated syngeneic female recipients, but only if the recipients were treated with testosterone. Testosterone treatment of donors did not affect blood glucose levels of recipients. Streptozotocin cytotoxicity in vitro determined by a 51Cr-release assay revealed an increased sensitivity to streptozotocin in dispersed islet cells from adult male animals as compared with cells from adult female mice. The incubation of islet cells from animals of either sex with testosterone, or oestradiol plus progesterone, did not enhance the susceptibility to streptozotocin. Islet cells from sexually immature male or female mice were less susceptible to streptozotocin. The results demonstrate that sex determines susceptibility to streptozotocin in vivo and in vitro.

HLA-D and -DR antigens in genetic analysis of insulin dependent diabetes mellitus.

Three groups of patients with insulin-dependent diabetes mellitus, ascertained by different procedures, were investigated for HLA-A, B, C and D antigens (n = 164), and a subset (n = 93) for HLA-DR. Both HLA-D/DR3 and D/DR4 were strongly positively associated and D/DR2 was negatively associated with insulin-dependent diabetes. HLA-DR+ was found to be a better marker for insulin-dependent diabetes than Dw4. The HLA-B associations (B8, B15 and B18) were clearly secondary to the increases of HLA-D/DR3 and D/DR 4. The HLA associations did not differ between familial and isolated cases indicating that these two groups may well have a common genetic background. Based on analysis of HLA-haplotype sharing in affected sibling pairs, a simple dominant model of inheritance could be ruled out, and a simple recessive model was found unlikely. The relative risks for the HLA-Dw3,4 and HLA-DR3,4 phenotype were 21.2 and 44.4 respectively and exceeded those of both the HLA-Dw3 and HLA-DR3 (5.6 and 4.3) as well as the HLA-Dw4 and DR4 (10.1 and 10.5) phenotypes. This argues against an intermediate genetic model but further studies are needed to clarify whether there is more than one susceptibility gene for insulin-dependent diabetes mellitus within the HLA-system.

Direct streptozotocin toxicity on dispersed mouse islet cells determined by [51]Cr-release.

Dispersed islet cells were prepared from collagenase-isolated lean mouse pancreatic islets by Dispase-II and subsequent mechanical treatment in calcium depleted media. An average yield of 600 cells per islet was obtained, 84% of the cells being beta-cells. Cells were incubated with radioactive chromium as a marker of cell viability. Optimal labelling of 1--2 cpm per cell was obtained by incubating 10(5) cells with 10(6) cpm of [51]Cr for 90 min. When islet cells were incubated with streptozotocin, this drug induced [51]Cr-release after a lag time of 2--4 hours. Furthermore, a positive correlation between streptozotocin concentrations and [51]Cr-release was found. This assay of cytotoxicity was highly reproducible and might be applicable in the study of other beta-cell damaging agents or autoimmune phenomena in the pathogenesis of diabetes.

Epidemiologic studies of insulin-dependent diabetes mellitus.

The incidence of insulin-dependent diabetes mellitus (IDDM) in Denmark in the years 1970--1976 was 13.3 per 100,000 in the age group 0--29 yr. This incidence is almost identical to that found in 1924 in Denmark in the same age group. The prevalence of insulin-consumers is 3.2 per 1,000. Comparison is made with incidence and prevalence data from other studies of Caucasian populations.

Studies of the HLA system and insulin-dependent diabetes mellitus.

The relationship between the HLA system and insulin-dependent diabetes mellitus (IDDM) is reviewed. Data compiled by the HLA and Disease Registry reveal that HLA-B8 and/or Dw3 are associated with IDDM in all populations studied so far, but further population studies in non-Caucasian populations should be performed. In Caucasians, HLA-Dw2 renders protection against IDDM while HLA-Dw3 and Dw4 are associated with susceptibility to IDDM. The exact mode of inheritance of susceptibility to IDDM remains to be established. Involvement of at least two genes is likely. Heterogeneity of IDDM is highly possible and should be a matter of major interest in diabetes research.

The influence of the major histocompatibility complex (H-2) on experimental diabetes in mice.

Mice with different histocompatibility loci on an identical background genome (congenic resistant lines of mice) were used to study the possible influence of the histocompatibility complex on experimental diabetes. The major histocompatibility complex (H-2) was not found to influence the diabetogenic effect of encephalomyocarditis (EMC) virus. In contrast the glucose intolerance following heterologous and homologous immunization with pancreatic antigens appeared H-2 influenced. Antibodies against cell surface components on viable B-cells were present in serum from mice with glucose intolerance induced by homologous immunization. The results suggest that the susceptibility to experimental autoimmune diabetes in mice is influenced by the H-2 complex.

Incidence of insulin-dependent diabetes mellitus (0--29 years at onset) in Denmark.

Incidence data from Denmark on the insulin-dependent diabetes mellitus in the age group 0--29 years have been collected in two different geographical areas (West and South Jutland plus Copenhagen and North Zealand). An incidence of 13.3 per 100,000 per year (range 12.5--13.6) was registered (N = 792). No difference was found between the two areas. The male incidence exceeded the female incidence by 25.4 per cent. The age distribution showed rising values until a peak in the early puberty with a decline until a rather constant level after puberty. From year to year a seasonal variation of onset was demonstrable with maximum in the winter and minimum in the summer for males only. Ascertainment was found to be between 87.7 and 98.6 per cent and the annual number of new insulin-dependent diabetics in Denmark in the age group 0--29 years can be calculated to 310-350.

HLA and insulin-dependent diabetes mellitus.

As suggested from clinical data and on the basis of human leukocyte antigen (HLA) data, insulin-dependent diabetes mellitus (IDDM) is a disease entity in itself and is different from non-insulin-dependent diabetes and other types of diabetes mellitus in aetiology and pathogenesis. HLA-B8 is associated with IDDM in all Caucasian populations studied, irrespective of the age of onset of the disease. HLA-B15 is associated with IDDM in populations of Northern European and British origin, while B18 seems to replace B15 in Southern European populations. IDDM is uncommon in populations where the HLA-B8 frequency is low, and in the Japanese IDDM occurs in association with Bw22. The HLA-Dw3 and Dw4 association with IDDM is stronger than that of the B alleles. Relative risks for B8 and B15 heterozygous and homozygous individuals are identical, i.e., no gene-dose effect exists. The relative risk of B8/B15 carriers is double that of relative risks of B8 and B15 alone, i.e., there are two IDDM-associated genes. The same applies to Dw3/Dw4 carriers. In families the phenotype IDDM segregates with a certain genotype, the diabetic proband's HLA haplotype. Only a small proportion of family members carrying the 'diabetic haplotype' develop IDDM.