Multiple roles of the DSCR1 (Adapt78 or RCAN1) gene and its protein product calcipressin 1 (or RCAN1) in disease.

The DSCR1 (Adapt78) gene is transiently induced by stresses to temporarily protect cells against further potentially lethal challenges. However, chronic expression of the DSCR1 (Adapt78) gene has now been implicated in several pathological conditions including Alzheimer's disease, Down syndrome and cardiac hypertrophy. Calcipressin 1 has been shown to function through direct binding and inhibition of the serine threonine protein phosphatase Calcineurin. Pharmacological inhibition of calcineurin, by the immunosuppressive drugs cyclosporin A and FK506, affects a wide variety of diseases. It is, therefore, likely that this endogenous calcineurin inhibitor, calcipressin 1, may also play a role in a variety of human diseases.

[Study of HRas1 minisatellite frequencies in children with thyroid papillary cancer]. / Izuchenie chastot minisatellita HRas1 sredi detei s papilliarnym rakom shchitovidnoi zhelezy.

The "rare" alleles of HRas1 gene minisatellite are well-known factor of predisposition to many kinds of cancer. We have studied HRas1 minisatellite frequencies among patients with papillary thyroid cancer which is related to consequences of Chernobyl accident. The HRas1 minisatellite was analysed in 32 patients who suffered from papillary carcinoma and underwent operation in 1996-2001 and in 75 Belorussian residents. Of 64 HRas1 alleles revealed in patients 14 were defined as "rare" (21.9%); in the control group we have detected 17 "rare" alleles (11.3%) of the examined 150 alleles. The higher frequency of "rare" HRas1 minisatellite alleles in patient group was statistically significant (p < 0.01). We can suppose that the "rare" alleles of HRas1 minisatellite are associated with increased risk of papillary thyroid cancer formation in children and adolescents after Chernobyl accident.

Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer's disease.

The DSCR1 (Adapt78) gene was independently discovered as a resident of the "Down syndrome candidate region"and as an "adaptive response"shock or stress gene that is transiently induced during oxidative stress. Recently the DSCR1 (Adapt78) gene product was discovered to be an inhibitor of the serine/threonine phosphatase, calcineurin, and its signaling pathways. We hypothesized that DSCR1 (Adapt78) might also be involved in the development of Alzheimer's disease. To address this question we first studied DSCR1 (Adapt78) in multiple human tissues and found significant expression in brain, spinal cord, kidney, liver, mammary gland, skeletal muscle, and heart. Within the brain DSCR1 (Adapt78) is predominantly expressed in neurons within the cerebral cortex, hippocampus, substantia nigra, thalamus, and medulla oblongata. When we compared DSCR1 (Adapt78) mRNA expression in post-mortem brain samples from Alzheimer's disease patients and individuals who had died with no Alzheimer's diagnosis, we found that DSCR1 (Adapt78) mRNA levels were about twice as high in age-matched Alzheimer's patients as in controls. DSCR1 (Adapt78) mRNA levels were actually three times higher in patients with extensive neurofibrillary tangles (a hallmark of Alzheimer's disease) than in controls. In comparison, post-mortem brain samples from Down syndrome patients (who suffer Alzheimer's symptoms) also exhibited DSCR1 (Adapt78) mRNA levels two to three times higher than controls. Using a cell culture model we discovered that the amyloid beta(1-42) peptide, which is a major component of senile plaques in Alzheimer's, can directly induce increased expression of DSCR1 (Adapt78). Our findings associate DSCR1 (Adapt78) with such major hallmarks of Alzheimer's disease as amyloid protein, senile plaques, and neurofibrillary tangles.

[ret/PtC1 and ret/PTC3r1 rearrangement in thyroid cancer cells, arising in residents of Belorus in the period after the accident at the Chernobyl nuclear power plant]. / Perestroiki ret/PTC1 i ret/PTC3r1 v kletkakh raka shchitovidnoi zhelezy, voznikshie u zhitelei Belorussii v period posle avarii na ChAES.

After the accident at the Chernobyl nuclear power plant, a considerable increase in the incidence of thyroid cancer among children in Belarus was observed. In the present study, the frequency of the c-ret protooncogene rearrangements in samples of thyroid carcinomas resected and diagnosed in 1998 from individuals in Belarus was investigated. The ret/PTC1 oncogene was detected in 19% of the samples, and the ret/PTC3r1 oncogene, in 14%. The number of ret/PTC1 rearrangements observed in tumor cells from the patients whose age at the time of the accident was from 1 to 10 years, was greater compared to those whose age at the time was from 10 to 20 years, irrespective of the year of surgery (1996 or 1998). The majority of the patients with ret/PTC3r1 rearrangements lived in Gomel oblast, which was contaminated by the Chernobyl meltdown.

Molecular alterations involving p53 codons 167 and 183 in papillary thyroid carcinomas from chernobyl-contaminated regions of belarus.

After the Chernobyl accident in 1986, there was a significant increase in the incidence of papillary thyroid carcinoma in fallout-exposed children from Belarus. We studied the p53 gene from 24 papillary thyroid carcinoma cases presenting in 1996. All subjects lived in contaminated regions of Belarus at the time of the accident and were under age 20 when exposed to fallout. Exons 5 through 9 of p53 were amplified from genomic tumor DNA using the polymerase chain reaction (PCR). The PCR products were analyzed by direct DNA sequencing using an automated sequencer. Five cases each exhibited two molecular alterations within exon 5. Alterations were confirmed by sequencing in both directions. One alteration, involving codon 167 (CAG-->CAT) in all five cases, resulted in the substitution of HIS for GLN. The second alteration, involving codon 183 (TCA-->TGA) in all five cases, resulted in a premature termination codon. Leukocyte DNA from each of the positive cases was analyzed and found to contain only wild-type p53 sequence. These results suggest that mutations involving codons 167 and 183 in the p53 locus are important in the pathogenesis of a subset (21%) of radiation-induced papillary thyroid carcinomas from Belarus.

[CD44 gene expression in cancerous thyroid cells]. / Ekspressiia gena CD44 v rakovykh kletkakh shchitovidnoi zhelezy.

The peculiarities of alternative CD44 mRNA splicing in thyroid cancer tissue of children from radiocontaminated areas was investigated. CD44 gene expression in thyroid cancer tissues of children exposed to radiation resembled that in spontaneously emerged cancers. It was concluded that CD44 gene expression is not the primary target of radioactive irradiation. Probably, the CD44 mRNA splicing deregulation is the consequence of cancer.

Expression of proopiomelanocortin (POMC)-derived melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) peptides in skin of basal cell carcinoma patients.

We proposed that local expression and production of proopiomelanocortin (POMC) peptides may play a role in human skin physiology and pathology, including the development and progression of skin cancers. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting hybridization techniques were used to study gene expression. Reversed-phase (RP) high-pressure liquid chromatography (HPLC) separation with subsequent radioimmunoassays were used to identify alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH) peptides. Immunocytochemistry (IHC) was used to localize ACTH, alpha-MSH, and beta-MSH antigens in skin. RT-PCR, RP-HPLC, and IHC analyses documented the expression of POMC mRNA and production of ACTH and alpha-MSH peptides in lesional and perilesional skin of basal cell carcinoma (BCC) patients and in cultured keratinocytes, which was accompanied by the expression of the MC1-R gene encoding the receptor activated by MSH and ACTH. Thirty specimens were analyzed by IHC. Immunoreactive alpha-MSH, beta-MSH, and ACTH were detected, in 21 of 21, in 11 of 20, and in 6 of 8 of lesional skin, and in 6 of 6, in 5 of 7, and in 6 of 8 perilesional skin specimens analyzed, respectively. Antigen distribution was heterogenous and present in BCC, epidermis, hair follicles, dermal mononuclear cells, and extracellular matrix. We conclude that messenger RNA for POMC, MC1-R, and the peptides MSH and ACTH are produced in skin of BCC patients. Because keratinocytes are a target for MSH and ACTH bioregulation, the production of these peptides is stimulated by UVB, and the peptides can act as immunosupressors, we suggest that MSH and ACTH may facilitate development of BCC.

Low prevalence of the ret/PTC3r1 rearrangement in a series of papillary thyroid carcinomas presenting in Belarus ten years post-Chernobyl.

After the Chernobyl accident in 1986, there was a significant increase in the incidence of papillary thyroid cancer in fallout-exposed children from Belarus. Radiation-induced rearrangements of chromosome 10 involving the c-ret proto-oncogene have been implicated in the pathogenesis of these cancers. The ret/PTC3r1 rearrangement was the most prevalent molecular lesion identified in post-Chernobyl papillary thyroid cancers arising in 1991 and 1992. We identified the ret/PTC1 rearrangement in 29% of 31 papillary thyroid cancers presenting in Belarus in 1996. In the present report, we examined 14 cases from this series (plus 1 additional case) and found a ret/PTC3r1 rearrangement in only 1 (7%). The prevalence of ret/PTC3r1 in this series is significantly lower than previously reported (p = 0.0006, Fisher exact test). This result suggests a switch in the ratio of ret/PTC3 to ret/PTC1 rearrangements in late (1996) versus early (1991-1992) post-Chernobyl papillary thyroid cancers.

The ret/PTC1 rearrangement is a common feature of Chernobyl-associated papillary thyroid carcinomas from Belarus.

An increase in the incidence of papillary thyroid cancer has been documented in individuals exposed to Chernobyl fallout in 1986. Experiments using cultured human cells have suggested that radiation can induce the ret/PTC1 rearrangement involving the ret proto-oncogene. To test the hypothesis that the ret/PTC1 rearrangement is involved in the pathogenesis of Chernobyl-associated papillary thyroid carcinomas, we studied a panel of 31 cases from Belarus. All individuals lived in fallout-contaminated oblasts (regions) of Belarus at the time of the accident: Gomel (n = 13), Brest (n = 12), Minsk (n = 4), and Grodno (n = 2). All were under age 20 at the time of the accident; 20 were born between 1982 and 1986. Individual thyroid radiation doses were estimated at 1.1 to 110 rem. Patients underwent surgery in Minsk in 1996. Fifteen patients had locally advanced disease (stage T4). The majority had regional lymph node involvement (stage N1, n = 27). There were no distant metastases. Surgical specimens were frozen at -80 degrees C, RNA was extracted and cDNA prepared. The polymerase chain reaction (PCR) was performed with specific primers for ret/PTC1, and c-ret and GAPDH as controls. Controls were positive in all 31 cases. Nine cases yielded a positive PCR product for the ret/PTC1 rearrangement (29%). Thus, the ret/PTC1 rearrangement is a feature of some Chernobyl-associated papillary thyroid cancers, and is one possible mechanism involved in the pathogenesis of these cancers.

Characterization of corticotropin-releasing hormone (CRH) in human skin.

We have confirmed the expression of CRH and CRH receptor type 1 genes in human skin, cultured HaCaT keratinocytes, squamous cell carcinoma, and melanoma cells. The size of CRH messenger ribonucleic acid (mRNA), estimated by Northern blot hybridization, was 1.5 kilobases. CRH peptide was identified by reverse phase high pressure liquid chromatography separation in both whole skin and cultured cells. Forskolin and dexamethasone at concentrations of 10 micromol/L stimulated and inhibited, respectively, CRH peptide production in squamous cell carcinoma and melanoma cells, but had no significant effect on the CRH mRNA level. In melanoma cells, stimulation of melanogenesis down-regulated CRH receptor type 1 mRNA expression, but was without effect on CRH mRNA production. We suggest that in human skin the CRH signaling system is both operative and under regulatory control.

Production of POMC, CRH-R1, MC1, and MC2 receptor mRNA and expression of tyrosinase gene in relation to hair cycle and dexamethasone treatment in the C57BL/6 mouse skin.

In skin of the C57BL/6 mouse, the production of mRNA transcripts that hybridized to the coding region of the MC1 receptor (MC1-R) gene was undetectable in telogen, increased during hair growth, and, after reaching the highest values in anagen VI, decreased during the anagen-catagen transition phase. This production was associated with anagen-dependent expression of the tyrosinase gene and enzyme activity. In contrast, the production of 4.5- and 2.0-kb mRNAs hybridizable to the coding region of the MC2 receptor (MC2-R) gene was similar throughout the entire hair cycle. Previously, dexamethasone was demonstrated to induce premature catagen development accompanied by an abrupt termination of melanogenesis. Here we demonstrate that topical application of dexamethasone during anagen VI decreased the concentration of POMC, MC1-R, and tyrosinase mRNA in the skin. The decrease in tyrosinase mRNA concentration was accompanied by a decrease in tyrosinase protein concentration and enzyme activity. These results support the hypothesis that murine hair growth and attendant melanogenesis can be regulated through coordinated changes in local expression of POMC, MC1-R, and tyrosinase genes.

Ultraviolet B stimulates production of corticotropin releasing factor (CRF) by human melanocytes.

Here we show that human melanocytes express the corticotropin releasing factor (CRF) gene and produce CRF peptide. The CRF production and secretion is markedly stimulated by ultraviolet B (UVB) radiation. This is the first demonstration that cutaneous melanocytes respond to environmental stress (UVB) through the production of CRF.

Novel CD44 messenger RNA isoforms in human thyroid and breast tissues feature unusual sequence rearrangements.

CD44 is a family of cell surface proteins implicated in adhesion interactions and tumor metastasis. Multiple CD44 mRNA isoforms arise from alternative splicing of variant exons (termed v1-v10). We recently discovered a novel CD44 mRNA isoform in human papillary thyroid cancers featuring a junction between subsegments of exons 4 and 13 (v8). The sequence ACAG was repeated at both the donor and acceptor sites in the genomic DNA (G. Ermak et al., Cancer Res., 56: 1037-1042, 1996). We used reverse transcription-PCR to characterize expression of this isoform in a panel of thyroid lesions. In addition, we assayed three cryopreserved human breast cancers and two samples of normal breast tissue (from female subjects who had undergone cosmetic mammoplasty) to determine whether a similar isoform is present in breast carcinomas. Levels of the novel isoform were up-regulated in 88% of the goiters, adenomas, and papillary cancers, but were undetectable in cases of thyroiditis and absent or low-level in four samples of normal thyroid tissue. The three breast cancers each yielded a 546-bp PCR product that was not detected in normal breast tissue. The PCR product from one of the breast cancers was cloned, and sequence analysis revealed a novel mRNA isoform featuring a junction between exon 3 and an internal site within exon 13 (v8). The sequence GCTTCAG was repeated at both the donor and acceptor sites in the genomic DNA. These results show that human thyroid and breast tissues contain novel CD44 mRNA isoforms featuring unusual rearrangements at repeated sequences. Further studies are warranted to determine whether the expression of this class of isoforms correlates with growth status.

Production and release of proopiomelanocortin (POMC) derived peptides by human melanocytes and keratinocytes in culture: regulation by ultraviolet B.

It is demonstrated that ultraviolet B (UVB) radiation stimulates increased expression of the proopiomelanocortin (POMC) gene which is accompanied by production and release of alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) by both normal and malignant human melanocytes and keratinocytes. The production and release of both peptides are also stimulated by dibutyryl cyclic adenosine monophosphate (dbcAMP) and interleukin 1 alpha (IL-1 alpha) but not by endothelin-1 (ET-1) or tumor necrosis factor-alpha (TNF-alpha). N-acetyl-cysteine (NAC), a precursor of glutathione (GSH), an intracellular free radical scavenger, abolishes the UVB-stimulated POMC peptide production and secretion. Conclusions are as follows: (1) Cultured human cells of cutaneous origin, namely keratinocytes and melanocytes, can produce and express POMC; (2) POMC expression is enhanced by exposure to UVB, possibly through a cyclic AMP-dependent pathway; and (3) The action of UVB on POMC production may involve a cellular response to oxidative stress.

ACTH receptor, CYP11A1, CYP17 and CYP21A2 genes are expressed in skin.

Evidence is provided that mRNA for ACTH (MC-2) receptor and mRNAs for three obligatory enzymes of steroid synthesis including cytochromes P450scc, P450c17 and P450c21 are expressed in normal and pathologic human skin. Thus, molecular elements of the distal loop of the "pituitary-adrenal axis" such as the MC-2, CYP11A1, CYP17 and CYP21A2 genes are expressed in the skin.

Metabolism of serotonin to N-acetylserotonin, melatonin, and 5-methoxytryptamine in hamster skin culture.

Biotransformation of [3H]serotonin by cultured hamster skin to 3H-metabolites corresponding to N-acetylserotonin (NAS), melatonin, and 5-methoxytryptamine (5-MT) was demonstrated. This process was time-dependent, with the highest production of radioactive NAS and melatonin metabolites after 3 and 5 h of incubation followed by a decrease in the rate of metabolite release into the media. Conversely, the formation of radioactive metabolite corresponding to 5-MT increased gradually during skin culture, reaching the highest level after 24 h of incubation. The production of 3H-metabolites, corresponding to NAS, melatonin, and 5-MT, was stimulated by forskolin with a maximum effect of forskolin at 10 microM concentration. The gas chromatographic/mass spectroscopy analysis of the fraction eluting at the retention time of NAS standard material showed that it contained NAS, further confirming production and release of NAS into the media by hamster skin. Therefore, we conclude that mammalian skin can acetylate serotonin to NAS and postulate that the NAS is further metabolized by the skin to form melatonin which is subsequently transformed to 5-MT.

The expression of proopiomelanocortin (POMC) and of corticotropin releasing hormone receptor (CRH-R) genes in mouse skin.

The proopiomelanocortin (POMC) and the corticotropin releasing hormone receptor (CRH-R) genes are expressed in the skin of the C57BL/6 mouse throughout the entire hair cycle, and CRH immunoreactivity is present in the pilosebaceous unit of the hair follicle and the epidermis. These findings suggest that some components of hypothalamo-pituitary axis are operating in mouse skin.

Restricted patterns of CD44 variant exon expression in human papillary thyroid carcinoma.

CD44 is a polymorphic family of cell surface proteoglycans and glycoproteins implicated in cell-cell and cell-matrix adhesion interactions, cell migration, and tumor metastasis. CD44 exists as a standard form and as multiple isoforms arising from alternative splicing of variant exons (termed v1-v10) encoding parts of the extracellular domain. We demonstrated previously that papillary thyroid carcinomas exhibit aberrant patterns of alternative CD44 mRNA splicing (G. Ermak et al., Cancer Res., 55: 4594-4598, 1995). In the present report, we use reverse transcription-PCR using a new high-performance polymerase formulation (Ex Taq; TaKaRa Shuzo Co., Ltd., Otsu, Japan) , followed by Southern hybridization, and demonstrate that alternative exon usage in papillary thyroid carcinomas is restricted primarily to exons v6, v7, v8, v9, and v10, with weak expression of v3. Expression of v8 is tightly linked to v9 and closely related to v10 expression. Also, v6 and v7 expression are closely related. Papillary thyroid cancers exhibit a marked increase in specific mRNA species containing combinations of exons v6 to v10. Several isoforms found in papillary cancers are not detectable in histologically normal tissue derived from the corresponding contralateral thyroid lobes. Examples include a 750-bp v6- and v7-containing PCR product and a 650-bp v8- and v9- containing PCR product. Finally, a novel 530-bp PCR product was discovered and shown to contain a subsegment from exon 4 joined to a subsegment of exon 13 (v8), followed by the complete sequence of exons 14 (v9) and 15 (v10). This novel isoform was present in both the papillary cancers and contralateral tissues. In conclusion, papillary thyroid cancers exhibit specific patterns of aberrant alternative CD44 splicing, distinguishing them from histologically normal thyroid tissue.