[Pharmacokinetics of n-(5-oxynicotinoyl)-L-glutamic acid calcium salt upon bolus administration in rats and rabbits: interspecies extrapolation].

The pharmacokinetics of N-(5-oxynicotinoyl)-L-glutamate (ONG) was studied in rats (doses, 20, 100 and 500 mg/kg) and rabbits (50 mg/kg) after bolus administration of calcium salt of N-(5-oxynicotinoyl)-L-glutamic acid (Ampasse preparation). The ONG concentration in the blood serum was determined by HPLC assay with fluorimetric detection. The lower limit of accurate detection for ONG was 100 ng/ml. The ONG pharmacokinetics in rats was linear at relatively small doses (20-100 mg/kg) but nonlinear at a large dose (500 mg/kg). The ONG concentration decay had a two-phase character in both rats and rabbits, so that the pharmacokinetic profiles were fitted to a biexponential equation of the two-compartment model. Systemic pharmacokinetic parameters determined in rats and rabbits, respectively, were as follows: total clearance, 18 and 15 ml/(min kg); steady state distribution volume, 330 and 880 ml/kg; mean retention time, 0.3 and 1.0 h; half-life, 0.73 and 2.3 h. Using the allometric approach to the interspecies extrapolation of the pharmacokinetic data, the half-life of ONG in humans is predicted to be 4 h.

Extracellular matrix regulates expression of the TGF-beta 1 gene.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell proliferation and modulates the interactions of cells with their extracellular matrix (ECM), in part by inducing the synthesis of various ECM proteins. Three different isoforms of TGF-beta are synthesized in a defined pattern in specific cell populations in vivo. In the specific case of TGF-beta 1, this well-defined and limited expression stands in sharp contrast to its synthesis by virtually all cells in culture. Using mammary epithelial cells as a model system, we evaluated the substratum dependence of the expression of TGF-beta 1. The level of TGF-beta 1 expression is high in cells on plastic, but is strongly downregulated when cells are cultured on a reconstituted basement membrane matrix. In contrast, TGF-beta 2 mRNA levels in cells on either substratum remain unchanged. Using the chloramphenicol acetyl transferase gene as reporter gene under the control of the TGF-beta 1 promoter, we show that transcription from this promoter is suppressed when the cells are in contact with either endogenously synthesized or exogenously administered basement membrane. TGF-beta 1 promoter activity is strongly induced by the absence of basement membrane, i.e., by direct contact of the cells with plastic. This modulation of transcription from the TGF-beta 1 promoter occurs in the absence of lactogenic hormones which allow full differentiation. Our results thus indicate that basement membrane is an important regulator of TGF-beta 1 synthesis, and explain why most cells in culture on plastic express TGF-beta 1 in contrast with the more restricted TGF-beta 1 synthesis in vivo. We propose that there is a feedback loop whereby TGF-beta 1-induced synthesis of basement membrane components is repressed once a functional basement membrane is present. Finally, these results together with our current knowledge of regulation of TGF-beta 1 and TGF-beta 2 synthesis, suggest that, in vivo, TGF-beta 1 may play a major role in regulating the ECM synthesis and the cell-ECM interactions, whereas TGF-beta 2 may be more important in morphogenetic processes.

Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo.

Members of the TGF-beta superfamily appear to modulate mesenchymal differentiation, including the processes of cartilage and bone formation. Nothing is yet known about the function of the TGF-beta-related factor vgr-1, also called bone morphogenetic protein-6 (BMP-6), and only limited studies have been conducted on the most closely related factors BMP-5, osteogenic protein-1 (OP-1) or BMP-7, and OP-2. Because vgr-1 mRNA has been localized in hypertrophic cartilage, this factor may play a vital role in endochondral bone formation. We developed antibodies to vgr-1, and documented that vgr-1 protein was expressed in hypertrophic cartilage of mice. To further characterize the role of this protein in bone differentiation, we generated CHO cells that overexpressed recombinant murine vgr-1 protein. Western blot analysis documented that recombinant vgr-1 protein was secreted into the media and was proteolytically processed to yield the mature vgr-1 molecule. To assess the biological activity of recombinant vgr-1 in vivo, we introduced the vgr-1-expressing CHO cells directly into the subcutaneous tissue of athymic nude mice. CHO-vgr-1 cells produced localized tumors, and the continuous secretion of vgr-1 resulted in tumors with a strikingly different gross and histological appearance as compared to the parental CHO cells. The tumors of control CHO cells were hemorrhagic, necrotic, and friable, whereas the CHO-vgr-1 tumors were dense, firm, and fibrotic. In contrast with control CHO tumors, the nests of CHO-vgr-1 tumor cells were surrounded by extensive connective tissue, which contained large regions of cartilage and bone. Further analysis indicated that secretion of vgr-1 from the transfected CHO tumor cells induced the surrounding host mesenchymal cells to develop along the endochondral bone pathway. These findings suggest that endochondral bone formation.

Aberrant expression of type I fibroblast growth factor receptor in human pancreatic adenocarcinomas.

Acidic and basic fibroblast growth factors are mitogenic polypeptides that are overexpressed in pancreatic cancer. To determine whether fibroblast growth factors may exert direct effects on pancreatic cancer cells in vivo, we compared the expression of the high-affinity type I fibroblast growth factor receptor (FGFR-1) in human pancreatic tissues. In the normal pancreas, FGFR-1 immunostaining was seen mainly in acinar cells. In pancreatic cancers, FGFR-1 was abundant in ductal-like cancer cells which also exhibited many FGFR-1 mRNA in situ hybridization grains. Analysis by the polymerase chain reaction and RNase protection revealed that the 2-immunoglobulin-like and the 3-immunoglobulin-like forms of FGFR-1 were expressed in all tissue samples, and that the 2-immunoglobulin-like form was overexpressed in the cancer tissues by comparison with the normal tissues. These findings suggest that the 2-immunoglobulin-like form of FGFR-1 may contribute to aberrant autocrine and paracrine pathways in pancreatic cancer.

Enhanced expression of the type II transforming growth factor beta receptor in human pancreatic cancer cells without alteration of type III receptor expression.

We have recently found that human pancreatic adenocarcinomas exhibit strong immunostaining for the three mammalian transforming growth factor beta (TGF-beta) isoforms. These important growth-regulating polypeptides bind to a number of proteins, including the type I TGF-beta receptor (T beta R-I), type II TGF-beta receptor (T beta R-II), and the type III TGF-beta receptor (T beta R-III). In the present study we sought to determine whether T beta R-II and T beta R-III expression is altered in pancreatic cancer. Northern blot analysis indicated that, by comparison with the normal pancreas, pancreatic adenocarcinomas exhibited a 4.6-fold increase (P < 0.01) in mRNA levels encoding T beta R-II. In contrast, mRNA levels encoding T beta R-III were not increased. In situ hybridization showed that T beta R-II mRNA was expressed in the majority of cancer cells, whereas mRNA grains encoding T beta R-III were detectable in only a few cancer cells and were present mainly in the surrounding stroma. These findings suggest that enhanced levels of T beta R-II may have a role in regulating human pancreatic cancer cell growth, while T beta R-III may function in the extracellular matrix.

Induction and expression of amphiregulin in human pancreatic cancer.

The epidermal growth factor receptor is activated by a family of polypeptides that includes the growth factor amphiregullin (AR). Using Northern blot analysis and the polymerase chain reaction, we now report that AR mRNA is expressed in human pancreatic cancer cell lines, and that this expression is enhanced in several of these cell lines by tetradecanoyl phorbol acetate and transforming growth factor alpha. AR was also expressed in normal and malignant pancreatic tissues. However, in the normal pancreas, AR immunostaining was most evident in the nuclei of ductal cells. In contrast, in many carcinomas, AR was also present in the cytoplasm of the ductal-like cancer cells. Cytoplasmic localization of AR was associated with a more advanced clinical stage. These findings suggest that AR may contribute to aberrant activation of the epidermal growth factor receptor in human pancreatic cancer, and may enhance disease progression.

Overexpression of acidic and basic fibroblast growth factors in human pancreatic cancer correlates with advanced tumor stage.

Acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) are mitogenic polypeptides that may contribute to cancer cell proliferation. In the present study we examined aFGF and bFGF expression in human pancreatic cancer. Northern blot analysis of total RNA isolated from 12 pancreatic cancers revealed elevated aFGF and bFGF mRNA levels in 12 and 10 samples, respectively, by comparison with the normal human pancreas. Immunostaining demonstrated the presence of aFGF and bFGF in many cancer cells and in the atrophic acini and ducts adjacent to the cancer cells, but to a much lesser extent in the surrounding fibroblasts. By in situ hybridization, both mRNA moieties colocalized with their respective proteins and were abundant in many cancer cells. Immunoblotting confirmed that cancer tissues with increased aFGF and bFGF immunoreactivity contained elevated levels of both proteins. To determine the significance of aFGF and bFGF expression in the pancreatic cancer cells, immunohistochemical analysis of 78 human pancreatic carcinomas was performed. aFGF and bFGF immunoreactivity was present in the cancer cells in 47 (60%) and 44 (56%) of the tumors, respectively. There was a significant correlation between the presence of either aFGF or bFGF in the cancer cells and advanced tumor stage, and the presence of bFGF and shorter patient survival. These data suggest that aFGF and bFGF are overexpressed in a significant proportion of human pancreatic carcinoma cells and that this overexpression may contribute to disease progression.

Enhanced erbB-3 expression in human pancreatic cancer correlates with tumor progression.

The erbB-3 gene encodes a transmembrane protein that is related to the epidermal growth factor (EGF) receptor and erbB-2. We compared erbB-3 expression in the normal human pancreas, human pancreatic carcinomas, and cultured human pancreatic cancer cell lines. Northern blot analysis of total RNA revealed the anticipated 6.2-kb mRNA transcript in all 19 normal pancreatic samples. In 17 of 27 pancreatic cancers, there was a 6.7-fold increase (P < 0.001) in erbB-3 mRNA levels. Southern blot analysis did not reveal erbB-3 gene amplification. Four of six pancreatic cancer cell lines exhibited the 6.2-kb erbB-3 mRNA transcript, and all four cell lines coexpressed the epidermal growth factor receptor and erbB-2. Using a highly specific antibody, we determined that faint to moderate erbB-3 immunoreactivity was present in the ductal cells in the normal pancreas. In 47% (27/58) of the pancreatic cancers, there were many cancer cells with intense erbB-3 immunostaining. The presence of erbB-3 in the cancer cells was associated with advanced tumor stage and shorter survival postoperatively. These data indicate that a significant proportion of human pancreatic cancers overexpress erbB-3, and that erbB-3 may contribute to disease progression in this disorder.

Transforming growth factor-alpha expression in the anterior pituitary gland: regulation by epidermal growth factor and phorbol ester in dispersed cells.

We have previously reported the immunohistochemical localization of transforming growth factor-alpha (TGF alpha) in the intact bovine anterior pituitary gland. Furthermore, we have purified TGF alpha from the conditioned medium of cell cultures derived from the bovine anterior pituitary. We report her the identification of the TGF alpha mRNA from both the intact bovine anterior pituitary gland and the anterior pituitary derived cell cultures. The level of TGF-alpha mRNA in the cell cultures is greater than that present in the intact gland. The TGF-alpha mRNA level increased when the cell cultures were allowed to incubate in their conditioned medium for 3 days, suggesting that a secretory product from the cultured cells is capable of stimulating the accumulation of the TGF-alpha mRNA. 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of these cells resulted in a 6-fold increase in the level of TGF-alpha secreted into the conditioned medium. TPA appears to stimulate TGF-alpha secretion at the level of gene transcription as TPA treatment also resulted in an increased accumulation of the TGF-alpha mRNA. The epidermal growth factor (EGF) receptor mRNA was examined in these cell cultures and it increased with TPA treatment in an analogous manner to the TGF-alpha mRNA. EGF treatment of the pituitary cells resulted in an increased level of TGF-alpha mRNA which followed the same time course as TPA, maximal stimulation occurred after 8 h of treatment. The magnitude of EGF stimulated TGF-alpha mRNA was not as great as that seen by TPA stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of epidermal growth factor-like mitogens by cultured cells from bovine anterior pituitary glands.

We have partially characterized growth factor activity secreted by early cultures of cells derived from bovine calf pituitary glands. Serum-free, chemically defined culture medium conditioned by pituitary cells stimulated DNA synthesis in rat kidney fibroblasts (NRK cells) equivalent to unconditioned medium containing 3 X 10(-10) M epidermal growth factor (EGF). The conditioned medium contained activity which competed with 125I-EGF for binding to EGF-receptors which was also equivalent to competition by 3 X 10(-10) M EGF in a radioreceptor assay. Secondary pituitary cultures produced about 10-fold more competing activity than did primary cultures and the production rate of the EGF-competing activity by the secondary cultures remained stable for over 4 weeks. Bio-Gel P-60 chromatography in 1 M acetic acid of an extract of conditioned medium resolved three peaks of EGF-competing activity. These peaks chromatographed with apparent mol wts of 17,000, 9,000, and 6,000. These peaks of activity were not as a result of EGF-binding proteins in the culture medium. The 6,000 mol wt peak contained activity which competed equally well with EGF for EGF receptor and antimouse EGF antibody binding. The 17,000 and 9,000 mol wt activities competed more effectively for binding to receptors than antibodies. The mitogenic activity in the three peaks correlated with the receptor-competing activity. However, the 17,000 and 9,000 mol wt peaks but not the 6,000 mol wt peak contained transforming growth factor activity capable of stimulating NRK cells to assume anchorage independent growth. These results suggest that a subpopulation of cells in pituitary glands secrete EGF and EGF-like transforming growth factors.

Alpha-transforming growth factor in the bovine anterior pituitary gland: secretion by dispersed cells and immunohistochemical localization.

A growth factor secreted by bovine calf anterior pituitary cells in culture was purified, and its N-terminal amino acid sequence was determined. This sequence shows near-identity with human and rat alpha-transforming growth factor (alpha TGF). With the use of an anti-alpha TGF monoclonal antibody generated against a C-terminal rat alpha TGF synthetic peptide, alpha TGF-like material was localized by immunohistochemical techniques in the cytoplasm of normal bovine adenohypophysial cells. The antibody staining was immunospecific because it could be completely inhibited by saturating concentrations of the synthetic peptide to which it was raised. There was no immunoreactivity in cells of the intermediate and posterior lobes. Some of the cells containing alpha TGF immunoreactivity also contained PRL; alpha TGF immunoreactivity was not demonstrated in cells containing ACTH, TSH, FSH, and LH. This is the first report documenting the secretion of alpha TGF by nonneoplastic adult cells and the presence of alpha TGF immunoreactivity in the corresponding normal adult tissue.

Binding characteristics of a biologically active variant of human growth hormone (20K) to growth hormone and lactogen receptors.

The dose-dependent displacement characteristics of a biologically active 20,000 molecular weight human pituitary growth hormone variant (20K) and of human growth hormone (hGH) were compared using hGH liver plasma membrane receptors both from 20-30 day pregnant rabbits and from normal female rats and also using mammary gland plasma membrane receptors from 5-6 day lactating rabbits. Different preparations of 20K were found to be only 3-20% as potent as hGH when compared at the dose necessary to cause 50% displacement of (125I)iodo-hGH from liver receptor and was 22-53% as effective as hGH in the mammary receptor assay system. These findings suggest that 20K and hGH may have separate receptors or that the binding characteristics of the two hormones may be quite different.

Ovarian transforming growth factor-alpha gene expression: immunohistochemical localization to the theca-interstitial cells.

Rat ovaries were examined for the presence of transforming growth factor-alpha (TGF-alpha). Immature diethylstilbestrol-primed rats were treated for 3 days prior to sacrifice with or without FSH. Their ovaries were either fixed in formaldehyde for immunohistochemistry or the RNA was extracted for northern blot analysis. Hybridization with a rat TGF-alpha cDNA probe revealed a 4.5 kilobase mRNA whose abundance was markedly stimulated in the rats treated with FSH. Immunohistochemical staining, using a sequence specific monoclonal antibody to TGF-alpha, MF9, detected immunoreactive TGF-alpha only in the interstitial and theca cells. FSH treatment resulted in no appreciable change in the immunostaining. These results suggest that TGF-alpha is synthesized in the ovary, perhaps in the theca-interstitial compartment.

p53 mutations are common in pancreatic cancer and are absent in chronic pancreatitis.

Pancreatic expression of the p53 tumor suppressor gene was studied in pancreatic adenocarcinomas and chronic pancreatitis. By immunohistochemistry, 16 of 34 (47%) cancers and none of the 24 chronic pancreatitis samples revealed nuclear staining. Sequence analysis indicated that 8 of 24 (33%) cancers were mutated for the p53 gene. Point substitutions occurred at codons 35, 105, 133, 213, 213, 258, and 299. A three base-pair in-frame insertion was identified between codons 261 and 262. None of 8 chronic pancreatitis samples exhibited p53 gene mutations. These data support a role for p53 gene alterations in human pancreatic cancer, and suggest that loss of its regulatory functions may constitute one of the differences between pancreatic cancer and chronic pancreatitis.

Increased mdm2 expression and immunoreactivity in human pancreatic ductal adenocarcinoma.

The murine double minute-2 (MDM2) gene encodes a 90 kDa protein which binds and inactivates the protein product of the p53 tumor suppressor gene. To elucidate the potential role of MDM2 in benign and malignant hyperproliferative conditions in the pancreas, we studied MDM2 expression in cultured human pancreatic cancer cells and in pancreatic tissues from normal donors, patients with pancreatic ductal adenocarcinoma and patients with chronic pancreatitis (CP). All the tested cell lines (PANC-1, COLO-357, HPAF and T3M4) expressed a 5.5 kilobase MDM2 mRNA transcript and exhibited nuclear MDM2 immunostaining. MDM2 mRNA levels were comparable in all 18 normal and 14 CP tissues for which RNA samples were available for analysis. By comparison with the normal samples, MDM2 mRNA levels were increased 6.4-fold in 8 of 12 human pancreatic cancer samples (p < 0.0001). All 8 samples exhibited nuclear MDM2 immunostaining, which was also present in 24 of 37 additional pancreatic cancers. Mild MDM2 immunoreactivity was seen in only 1 of 20 CP samples and in none of the 18 normal samples. These findings indicate that MDM2 is overexpressed in a majority of pancreatic adenocarcinomas, but not in CP samples. This selective overexpression raises the possibility that MDM2 may contribute to pancreatic neoplastic transformation by interfering with the growth-suppressing activity of wild-type p53.

Betacellulin, a member of the epidermal growth-factor family, is overexpressed in human pancreatic-cancer.

Human pancreatic ductal adenocarcinomas overexpress the epidermal growth factor (EGF) receptor. Betacellulin is a mitogenic polypeptide that binds and activates this receptor. To determine whether betacellulin has a role in human pancreatic cancer, we studied its expression in cultured human pancreatic cancer cell lines and in normal and cancerous pancreatic tissues. Five of 6 pancreatic cancer cell lines expressed the 3 kb betacellulin mRNA moiety, T3M4, MiaPaCa-2 and COLO-357 cells exhibiting the highest expression levels. EGF, heparin-binding EGF-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF) increased betacelullin mRNA levels. Only 2 of 15 normal samples and 1 of 10 cancer samples failed to exhibit the betacellulin transcript. Densitometric analysis of the autoradiographs revealed a 7.5-fold increase in betacellulin mRNA levels in the cancer tissues by comparison with the normal tissues. By in situ hybridization, the duct-like cancer cells exhibited many betacellulin mRNA in situ hybridization grains. These findings indicate that human pancreatic cancer cells express betacellulin in culture and in vivo, and suggest that this EGF-like ligand may participate in aberrant autocrine and paracrine activation of the EGF receptor in human pancreatic cancer.

Transforming growth factor-alpha in normal and neoplastic human endocrine tissues.

Transforming growth factor-alpha (TGF-alpha) is a polypeptide growth factor that binds to the epidermal growth factor receptor and is though to stimulate cell proliferation. It has been believed to play a role in tumor initiation by inducing the reversible transformed phenotype; overexpression of TGF-alpha may be important for tumor progression via autocrine stimulation and oncogene overexpression. Expression of TGF-alpha and the epidermal growth factor receptor has been documented in several nontumorous tissues and in a variety of tumors. This study used immunohistochemistry to localize TGF-alpha expression in normal and neoplastic endocrine tissues. Transforming growth factor-alpha was found in nontumorous hypothalamus, pituitary, thyroid, parathyroid, adrenal cortex and medulla, and pancreatic islets. Immunoreactivity was detected in most benign and malignant tumors of these tissues, as well as in endocrine neoplasms of the lung and gastrointestinal tract. Hypothalamic gangliocytomas, pheochromocytomas, and adrenal cortical carcinomas showed consistently greater immunoreactivity than their normal counterpart, but there was no correlation between degree of reactivity and tumor grade, stage, or hormone content. These results suggest that in endocrine tissues, TGF-alpha is unlikely to prove useful as a tumor marker but that the growth factor may play a role in both normal physiology and tumorigenesis.

Overexpression of HER2/neu oncogene in human pancreatic carcinoma.

The HER2/neu oncogene encodes a transmembrane protein that possesses intrinsic tyrosine kinase activity. Its overexpression has been associated with the malignant phenotype. In this study we examined HER2/neu expression in the normal and cancerous human pancreas. In the normal pancreas HER2/neu immunostaining was observed in acinar and ductal cells. HER2/neu immunoreactivity was expressed in 34 of 76 (45%) pancreatic carcinomas. There was a significant correlation between tumors with well-differentiated histology and HER2/neu expression. Northern blot analysis demonstrated HER2/neu mRNA expression in the normal pancreas and in situ hybridization confirmed its distribution in both acinar and ductal cells. In cancer tissues Northern blot analysis indicated that HER2/neu mRNA levels were elevated in 13 of 25 (52%) of the tumors in comparison with the normal tissues. In addition, in situ hybridization demonstrated a strong but heterogenous distribution of mRNA grains in these tumors. Southern blot analysis did not demonstrate HER2/neu gene amplification in any of the tumors. These data indicate that the HER2/neu protein is synthesized in the normal exocrine pancreas and is frequently overexpressed in well-differentiated adenocarcinomas of the pancreas as a result of increased HER2/neu mRNA levels.

Single-strand conformation polymorphism analysis of the epidermal growth factor receptor at codon 497.

A variant human epidermal growth factor (EGF) receptor (HER) with a transition (G to A) at codon 497, resulting in a substitution of a lysine for an arginine, was recently demonstrated in several human pancreatic cancer cell lines. In the present study, we compared the frequency of expression of wild-type (HER497R) and variant (HER497K) EGF receptors in normal and malignant pancreatic tissue samples using single-strand conformation polymorphism analysis. Both receptors were expressed in normal (42%) and malignant (46%) tissues. Three samples (two normal and one cancer) expressed only HER497K. The cancer sample that was homozygous for HER497K exhibited strong EGF receptor immunoreactivity. The high frequency of HER497K expression suggests that it is due to a relatively common polymorphism in the HER coding region. Its presence in some of the cancer samples suggests that, like the wild-type receptor, HER497K also has a role in pancreatic cancer.

Cytotoxic effects of TGF-alpha-Pseudomonas exotoxin A fusion protein in human pancreatic carcinoma cells.

The epidermal growth factor (EGF) receptor is overexpressed in human pancreatic cancers and cultured cell lines. TP40 is a chimeric protein composed of transforming growth factor-alpha (TGF-alpha) linked to a modified Pseudomonas exotoxin A (PE40) that exerts growth inhibitory effects on cells bearing a high number of EGF receptors. Therefore, we compared the effect of TP40 on the growth of Chinese hamster ovary (CHO), cells expressing varying levels of the EGF receptor and on the growth of two human pancreatic cancer cell lines. The growth of CHO cells devoid of endogenous EGF receptors was minimally altered by high concentrations of TP40, even following a 72-h incubation period. In contrast, in CHO cells expressing approximately 95,000 and 438,000 EGF receptors per cell, one-half maximal growth inhibition occurred at 5 and 3 ng/ml TP40, respectively. Following a 72-h incubation in T3M4 and COLO 357 human pancreatic cancer cells, one-half maximal growth inhibition occurred at 0.2 and 0.4 ng/ml TP40, respectively. This effect was significantly greater than that of native Pseudomonas exotoxin A. These findings indicate that human pancreatic cancer cells are markedly sensitive to the growth inhibitory effects of TP40 and raise the possibility that TP40 may have a therapeutic role in this disorder.