Calretinin is essential for mesothelioma cell growth/survival in vitro: a potential new target for malignant mesothelioma therapy?

Malignant mesothelioma (MM) are highly aggressive asbestos-related neoplasms, which show strong chemotherapy resistance, and there is no effective cure for MM so far. Calretinin (CR) is widely used as a diagnostic marker for epithelioid and mixed (biphasic) mesothelioma; however, little is known about CR's putative functions in tumorigenesis. CR protects against asbestos-induced acute cytotoxicity mediated by the AKT/PI3K pathway, and furthermore, SV40 early region genes are able to upregulate CR in mesothelial cells. However, the precise role of CR in mesothelioma is still unknown. Downregulation of CR via lentiviral-mediated short-hairpin RNA significantly decreased the viability and proliferation of mesothelioma cells in vitro. The effect was strong in epithelioid-dominated cell lines (ZL55 and MSTO-211H). A weaker and delayed effect was observed in mesothelioma cells with prevalent sarcomatoid morphology (SPC111, SPC212 and ZL34). The specificity of the effect was confirmed by stable enhanced green fluorescent protein-CR expression in mesothelioma cell lines and subsequent downregulation. Depletion of CR led these cancer cell lines to enter apoptosis within 72 hr postinfection via strong activation of the intrinsic caspase 9-dependent pathway. Downregulation of CR in immortalized mesothelial cells LP9/TERT-1 strongly blocked proliferation and caused a G1 block without decreasing viability or activating apoptosis pathways. These results demonstrate that downregulation of CR had a strong effect on the viability of MM cells and that CR is essential for cells derived from MM. The authors anticipate these findings to reveal CR as a highly interesting new putative therapeutic target for mesothelioma treatment of especially the epithelioid, as well as of the mixed and sarcomatoid type.

Changes in the distribution of calbindin D28-k, parvalbumin, and calretinin in the hippocampus of the circling mouse.

The circling (cir) mouse strain, a murine model of deafness caused by a spontaneous mutation, exhibits characteristic behaviors of circling and hyperactivity. In an induced-noise paradigm, cir mice display a significant loss in their spatial orientation abilities, and this has been suggested to be due at least in part to changes in calcium homeostasis. Auditory information is transferred from the cochlear nucleus to the hippocampus, where it is processed to modulate motor and sensory activity. Such a pathway could be affected at the cellular level by alterations in neurotransmission, including alterations that involve Ca(2+). However, there have been no studies in a hearing deficit model examining the concomitant molecular alterations in the hippocampus. Thus, in the present study we used immunohistochemistry to compare the distribution of the calcium-binding proteins (CaBPs) calbindin D-28k, parvalbumin, and calretinin in the hippocampi of heterozygous (+/cir), homozygous (cir/cir), and wild-type (+/+) mice. The expression of the CaBPs in various hippocampal subfields appeared to be significantly lower in cir mice (+/- and -/-) than in +/+ mice. Such a decrease in CaBP expression in cir/cir mice would alter calcium homeostasis, which in turn could affect the connection of the tri-synaptic circuit of the hippocampus as well as the cortical region. A decrease in CaBPs and the probable resultant glutamate-mediated excitability could contribute to the functional changes that lead to the characteristic behavioral features of cir mice.

Decrease of calbindin-d28k, calretinin, and parvalbumin by taurine treatment does not induce a major susceptibility to kainic acid.

Taurine, 2-aminoethanesulfonic acid, is present at high concentrations in many invertebrate and vertebrate systems, and it has several biological functions. In addition, it has been related to a neuroprotective role against several diseases, such as epilepsy. It has been reported that taurine induces a decrease of calbindin-D28k, calretinin, and parvalbumin protein levels in the hippocampus 3 days after administration. In the present work we hypothesized that the decrease of these proteins could alter the action of kainic acid (KA) and make mice more susceptible to excitotoxicity. Therefore, we treated mice with taurine and after 3 days treated them with KA. The results showed that taurine pretreatment did not induce a major susceptibility to KA. Moreover, neurodegeneration was reduced in pretreated mice. However, astrogliosis was similar to that observed in mice treated only with KA. The immunohistochemistries for calbindin-D28k, calretinin, and parvalbumin showed that these proteins were reduced as a consequence of KA treatment and of taurine treatment. However, mice pretreated with taurine prior to KA administration presented the same reduction in these proteins as mice treated with only taurine or only KA.

Immunohistochemical localization of calbindin D28-k, parvalbumin, and calretinin in the cerebellar cortex of the circling mouse.

The spontaneous mutant circling mouse has an autosomal recessive pattern of inheritance and is an animal model for deafness, which is characterized by circling, head tossing, and hyperactivity. Since the main pathology in circling mice lies in the organ of Corti, most studies on deaf mice have focused on auditory brain stem nuclei. No studies regarding behavior-related CNS changes in circling mice have been reported. The major center of sensory input for modulation of motor activity is best-studied in the cerebellum. Considering the importance of calcium homeostasis in numerous processes, calcium-binding proteins (CaBPs), such as calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR), may play crucial roles in preserving cerebellar coordinated motor function. Thus, the distribution of CB, PV, and CR was determined in the cerebellum using immunohistochemical methods to compare immunoreactivity (IR) of CaBPs between wild-type (+/+), heterozygous (+/cir), and homozygous (cir/cir) mice. The IR of CB and PV was predominantly observed in the Purkinje cell layer of all three genotypes. Compared with the +/+ genotype, the relative mean density of CB and PV IR in the Purkinje cell layer and CR IR in the granular layer was significantly decreased in the cir/cir genotype. Changes in calcium homeostasis in parallel fiber/Purkinje cell synapses could diminish cerebellar control of motor coordination. A number of deficiencies among the CaBPs lead to distinct alterations in brain physiology, which may affect normal behavior.

Aluminum toxicity alters the regulation of calbindin-D28k protein and mRNA expression in chick intestine.

Previous studies have shown that aluminum inhibits vitamin D-dependent calcium absorption. The mechanism involves reduced sensitivity to 1,25-dihydroxycholecalciferol and reduced expression of the calcium transport protein, calbindin-D28k. Reduced expression of calbindin protein may be due to decreased levels of calbindin mRNA. To test this hypothesis, we measured calbindin mRNA levels in chicks fed diets with and without added aluminum. Groups of chicks were fed one of four diets: control, control plus aluminum, low calcium, or low calcium plus aluminum. A fifth group was fed a vitamin D-free diet as a negative control. Calbindin protein was measured by immunoblotting. Serum calcium and inorganic phosphorus were determined. Intestinal mRNA was isolated and assayed by slot-blot hybridization to a fluorescein-conjugated oligonucleotide probe complementary to calbindin-D28k mRNA. Antifluorescein antibodies conjugated to alkaline phosphatase were used to detect hybrids and mRNA levels were quantified by densitometry. Specificity of the probe was verified by Northern analysis. Intestinal calbindin protein was greater in the control plus aluminum group than in controls, but no difference in calbindin mRNA was observed. These changes were associated with small decreases in serum phosphorus and calcium, suggesting a postranscriptional effect of aluminum. Chicks fed the low calcium diet had greater intestinal calbindin protein and mRNA levels relative to the control group in association with a 45% decrease in serum calcium. In contrast, no difference in calbindin protein, and significantly less mRNA were found in the low calcium plus aluminum group compared with controls, despite a decrease in serum calcium similar to that of chicks fed the low calcium diet without aluminum. These results show that in chicks fed a low calcium diet, aluminum intake decreases transcription and/or stability of intestinal calbindin mRNA, and that aluminum may inhibit the expression of vitamin D-dependent genes.

The effects of calbindin D-28K and parvalbumin antisense oligonucleotides on the survival of cultured Purkinje cells.

The role of calcium binding proteins, calbindin D-28k (CaB) and parvalbumin (PV) in Purkinje cell survival was investigated using oligonucleotide antisense strategy. Purkinje cell enriched cultures were prepared from the cerebella of 0-1 day old Balb/c mouse pups. Purkinje cells were identified by size, asymmetric arbors, immunoreactivity to CaB and PV, uptake of gamma-aminobutyric acid (GABA) and failure to express glial fibrillary acidic protein. The cells at different days in vitro were treated with antisense or mismatched antisense phosphorothioate oligonucleotides for CaB and PV mRNA (complexed with lipofectin). Neuronal specific [3H]-GABA uptake was used as a measure of Purkinje cell survival. The cultures treated for 24 h with antisense oligos (CaB+PV) showed a significant decrease in [3H]-GABA uptake as compared with the cultures treated with lipofectin alone or with lipofectin + mismatched antisense oligos to CaB and PV mRNA. The results of the present study suggest that the expression of calcium buffering proteins CaB and PV may have a significant involvement in Purkinje cell viability.

Calmodulin-like effect of oncomodulin on cell proliferation.

Oncomodulin (OM) is a Ca2+ binding protein (CABP) structurally closely related to parvalbumin. Expression of OM is restricted to early embryonic stages, the placental cytotrophoblasts, and neoplastic tissues. The function of OM as a calmodulin (CaM)-like enzyme modulator is controversial. Two types of experiments demonstrate that OM may act in an analogous fashion to CaM in T14 and T10 cancerous cell lines, which both express OM. First, both OM transcript and protein levels increased at the G1/S boundary in a similar manner to CaM, though not to the same extent, in the chemically transformed rat fibroblast cell line T14 synchronized at mitosis by nocodazole. Second, antisense oligonucleotides specific to the OM ATG region inhibited growth of T14 in a similar dose-dependent manner as observed with CaM-specific antisense probes.