Outcome improvement with chemotherapy and radiotherapy in primary, localized, radiation-associated angiosarcoma of the breast region: a retrospective case series analysis.

BACKGROUND: We report on a series of consecutive patients with localized radiation-associated angiosarcoma (RAAS) of the breast region (BR) treated at two Italian sarcoma reference centers. MATERIALS AND METHODS: We retrospectively reviewed all cases of primary, localized, resectable RAAS of the BR, treated at one of the two participating institutions from 2000 to 2019. Relapse-free survival (RFS) and overall survival (OS) were calculated. The prognostic role of several variables was investigated. A propensity score matched (PSM) analysis was carried out. RESULTS: Eighty-four patients were retrospectively identified. Nineteen out of 84 patients (22.6%) were pretreated with an anthracycline-based regimen for previous cancer. All patients but one underwent surgery, with 37/84 (44.1%) receiving surgery alone and 46/84 (54.8%) a multimodal approach: 18/84 (21.4%) received radiation therapy (RT) and 46/84 (54.9%) received chemotherapy. An anthracycline-based regimen was used in 10/84 patients (11.9%), while a gemcitabine-based regimen was used in 33/84 (39.3%). With a median follow-up of 51 months (interquartile range: 30-126 months), 36/84 patients (42.9%) relapsed and 35/84 patients (41.7%) died (8/84, 9.5% in the lack of metastatic disease). Five-year OS and 5-year RFS were 57% [95% confidence interval (CI) 43% to 68%] and 52% (95% CI 39% to 63%), respectively. Both (neo)adjuvant RT and chemotherapy were associated with better RFS [hazard ratio (HR) 0.25, 95% CI 0.08-0.83; HR 0.45, 95% CI 0.23-0.89] with a trend towards a better OS (HR 0.51, 95% CI 0.18-1.46; HR 0.60, 95% CI 0.29-1.24). Gemcitabine-based regimens seemed to perform better (HR 4.28, 95% CI 1.29-14.14). PSM analysis retained the above results. CONCLUSIONS: This retrospective study supports the use of (neo)adjuvant RT and chemotherapy, in primary, localized resectable RAAS of the BR. An effort to prospectively validate the role of (neo)adjuvant RT and chemotherapy is warranted.

Evaluation of an individualized education program in pituitary diseases: a pilot study.

INTRODUCTION: The low prevalence of pituitary diseases makes patient autonomy crucial, and self-management programs should be more common. OBJECTIVES: To assess the efficacy of an education program for patients with pituitary diseases in terms of patients' quality of life, satisfaction and goal attainment. DESIGN AND METHODS: Adult patients with pituitary disorders were recruited in a tertiary referral center and chose at least three of eight possible sessions on various topics, from disease management to psychosocial issues. Patients were included if they attended at least three sessions between 2012 and 2016 and completed the initial, final, and follow-up questionnaires. Data on quality of life (SF36), satisfaction and goal attainment were analyzed. RESULTS: Fifty-three patients were included (33 women; mean age, 53.5 years). There were a significant quality of life improvements in terms of physical and psychic limitation scores at the final assessment that persisted at follow-up evaluation. Most patients reached their objectives, especially those on sharing experiences and improving autonomy and self-confidence. More than half set new objectives at the end of the program, the most popular one being to reinforce their knowledge of their pituitary disease, its evolution and treatment (17.1% of patients). The mean overall satisfaction score was 3.75/4. At follow-up evaluation, patients reported improved self-management of pituitary disease (3.6/5) and improved self-efficacy (3.8/5). CONCLUSION: Individualizing the educational objectives of patients with pituitary disease improves the way they live with their disease. If confirmed in other cohorts, this approach could become the gold standard for education programs in rare endocrine diseases.

[How I manage... Malignant pleural mesothelioma in 2019]. / Comment je traite Le mésothéliome pleural malin en 2019.

Malignant pleural mesothelioma is a rare disease originating from mesothelial cells of the pleura and is related to asbestos exposure. The tumor is generally extended at the time of diagnosis and the treatment consists of a systemic palliative therapy. Radical approach is limited to very selected patients and is performed in expert centers but without validated schema. Radiotherapy alone is mainly used in palliative intent. Platinum-based chemotherapy in association with pemetrexed is the frontline standard of care and provides a 12-month overall survival. The addition of bevacizumab, an antiangiogenic drug, shows an improvement in median survival. To date, there is no second-line treatment approved for this disease and therefore inclusion in trials is recommended. Currently, various studies are investigating target therapy, immunotherapy and intrapleural perioperative treatment.

Le mésothéliome pleural malin est une tumeur rare, issue des cellules mésothéliales de la plèvre et liée à un contact avec l'amiante. Au moment du diagnostic, la maladie est souvent de stade avancé et est prise en charge par un traitement systémique palliatif. Un traitement radical est réservé pour de rares cas très sélectionnés, au sein de centres experts et ce, sans qu'aucun schéma de prise en charge ne soit validé. La radiothérapie seule est essentiellement utilisée à titre palliatif antalgique. Le traitement systémique de référence consiste en une chimiothérapie à base de cisplatine et pemetrexed permettant une survie globale de 12 mois. L'ajout à la chimiothérapie d'une thérapie ciblée anti-angiogénique, le bévacizumab, a permis une amélioration significative de la survie. A ce jour, il n'y a pas de traitement de 2ème ligne validé et il est donc recommandé d'inclure les patients dans des études cliniques. Actuellement, de multiples études évaluent des thérapies ciblées, des immunothérapies et des traitements intrapleuraux peropératoires.

[Invasive diagnostic techniques for diffuse interstitial pneumopathies]. / Comment j'explore... Techniques diagnostiques invasives des pneumopathies interstitielles diffuses.

Interstitial lung diseases represent a very heterogeneous group of diseases mainly affecting connective lung tissue even if alveolar space may sometimes be involved. The identification of their etiology is the key stage in their management. It requires the integration of anamnestic, clinical, biological, radiological data and, sometimes relies on, cytology or histology. In this review, we assess the contribution and feasibility of the different invasive techniques used for interstitial lung disease diagnosis. In particular we focus on the yield of lung endoscopy in casting light on the multidisciplinary confrontation, which is the gold standard of the interstitial lung disease care management.

Les pneumopathies interstitielles diffuses constituent un groupe très hétérogène de pathologies respiratoires qui affectent le parenchyme pulmonaire et qui se manifestent radiologiquement par des opacités interstitielles, même si une atteinte alvéolaire peut y être associée. L'identification de leur étiopathologie constitue une étape clé dans leur prise en charge thérapeutique. Elle nécessite l'intégration de données anamnestiques, cliniques, biologiques, radiologiques et parfois cyto/histologiques. Le but de cette revue est de préciser l'apport respectif et la faisabilité des diverses techniques semi-invasives et invasives d'exploration d'une pneumopathie interstitielle diffuse. En particulier, l'endoscopie pulmonaire fournit des éléments qui permettent d'éclairer la confrontation multidisciplinaire, cette dernière étant le gold standard de la prise en charge de ces pneumopathies.

Tailored chemotherapy based on tumour gene expression analysis: breast cancer patients' misinterpretations and positive attitudes.

The aim of this study was to document how breast cancer patients perceive their prognosis and a tailored treatment based on tumour gene expression analysis, and to identify the features of this approach that may impact its clinical application. In-depth interviews were conducted at three French cancer centres with 37 women (35-69 years of age) with node-positive breast cancer undergoing an adjuvant chemotherapy regimen defined on the basis of the genomic signature predicting the outcome after chemotherapy. Several concerns were identified. First, some misconceptions about these methods were identified due to semantic confusions between the terms 'genomic' and 'genetic', which generated anxiety and uncertainty about the future. Second, the 'not done' and 'not interpretable' signatures were misinterpreted by the women and associated with highly negative connotations. However, the use of tumour genomic analysis to adapt the treatment to each patient received most of the patients' approval because it was perceived as an approach facilitating personalised medicine. In conclusion, improving the quality of provider/patient communications should enable patients to play a more active part in the decision making about their treatment. This will ensure that those who agree to have tumour gene analysis have realistic expectations and sound deductions about the final result disclosure process.

Pituitary transcription factors: from congenital deficiencies to gene therapy.

Despite the existence of interspecies phenotypic variability, animal models have yielded valuable insights into human pituitary diseases. Studies on Snell and Jackson mice known to have growth hormone, prolactin and thyroid-stimulating hormone deficiencies involving the hypoplastic pituitary gland have led to identifying alterations of the pituitary specific POU homeodomain Pit-1 transcription factor gene. The human phenotype associated with rare mutations in this gene was found to be similar to that of these mice mutants. Terminal differentiation of lactotroph cells and direct regulation of the prolactin gene both require interactions between Pit-1 and cell type specific partners, including panpituitary transcriptional regulators such as Pitx1 and Pitx2. Synergistic activation of the prolactin promoter by Pitx factors and Pit-1 is involved not only in basal condition, but also in responsiveness to forskolin, thyrotrophin-releasing-hormone and epidermal growth factor. In corticotroph cells, Pitx1 interacts with Tpit. Tpit mutations have turned out to be the main molecular cause of neonatal isolated adrenocorticotrophin deficiency. This finding supports the idea that Tpit plays an essential role in the differentiation of the pro-opiomelanocortin pituitary lineage. The effects of Pit-1 are not restricted to hormone gene regulation because this factor also contributes to cell division and protects the cell from programmed cell death. Lentiviral vectors expressing a Pit-1 dominant negative mutant induced time- and dose-dependent cell death in somatotroph and lactotroph adenomas in vitro. Gene transfer by lentiviral vectors should provide a promising step towards developing an efficient specific therapeutic approach by which a gene therapy programme for treating human pituitary adenomas could be based.

Expression of peptide-23/pancreatitis-associated protein and Reg genes in human pituitary and adenomas: comparison with other fetal and adult human tissues.

Peptide 23, the rat homolog of the human pancreatitis-associated protein (PAP)/hepatocarcinoma-intestine-pancreas (HIP) protein, has been identified in primary culture of rat pituitary cells. Its secretion was shown to be stimulated by GH-releasing factor and inhibited by somatostatin in a similar fashion to GH. This observation led the researchers to speculate that peptide 23 does have a physiological hormonal role. We tested this hypothesis by screening by RT-PCR reactions the expression of the PAP/HIP gene in several human pituitary adenomas, especially GH-producing adenomas. Our results show a weak expression of the PAP/HIP gene in the pituitary gland and in most of the tumors, but independent of their origin. The significant homology of the PAP/HIP gene to the Reg gene family prompted us to study in the same pituitary adenomas the presence of the related Reg genes. Reg expression was never observed in the adenomas tested or in the pituitary gland. In contrast, the RegL transcript was observed in pituitary gland and in some subtypes of adenomas. We then extended our work to normal adults and developing human tissues to compare the expression patterns of the PAP/Reg gene family. We observed the presence of the PAP/HIP transcript in each tissue tested. In contrast, the Reg gene was expressed only in fetal pancreas and in some adult tissues, whereas the RegL gene was expressed not only in fetal pancreas but also in fetal colon and brain as well as some adult tissues. In conclusion, our results show that all of the human fetal and adult tissues examined express at least one of the different transcripts of the PAP/Reg family, suggesting that the regulation of these homologous genes is coordinately controlled.

Prolactinomas resistant to bromocriptine: long-term efficacy of quinagolide and outcome of pregnancy.

Resistance to bromocriptine, defined as the absence of normalization of prolactin (PRL) levels despite a 15-30 mg daily dose of bromocriptine during at least 6 months, has been observed in 5-17% of the prolactinomas according to the literature. The recent availability of a new potent dopamine agonist, quinagolide, prompted us to analyze its long-term therapeutic effects in 28 patients with prolactinomas resistant to bromocriptine. Before bromocriptine, their PRL levels were 520 +/- 185 micrograms/l (mean +/- SEM) and decreased to 291 +/- 154 micrograms/l after a 6-21 month period of bromocriptine treatment. All the women (N = 20) remained amenorrheic and hypogonadism was not improved in men (N = 8). Subsequently, after 1 year of 150-300 micrograms/day quinagolide, 12/28 patients of the present series recovered normal gonadal function and their initial mean baseline PRL value (404 +/- 180 micrograms/l) was 16 +/- 2 micrograms/l after 1 year of treatment. A significant tumor shrinkage was observed in 5/8 macroadenomas (62%). During the 3-year follow-up period under quinagolide, a similar good control was achieved in these patients, with the exception of one man presenting with a secondary rise of PRL under quinagolide. In contrast, 15 other patients (one patient interrupted quinagolide at 6 months because of poor tolerance) were not normalized under 150-450 micrograms/day quinagolide. Their initial PRL levels (606 +/- 298 micrograms/l) were reduced to 343 +/- 187 micrograms/l (versus 463 +/- 265 micrograms/l under bromocriptine after the same duration of treatment). Despite such a partial inhibitory effect of quinagolide, 7/12 women resumed menstrual cycles and three pregnancies occurred. In no case was any tumor shrinkage noticed during the 3-4-year follow-up. Three patients even presented, after 2 years of quinagolide treatment, with a secondary rise of PRL values associated with a further tumor growth in two patients. During the 3-year follow-up period, nine pregnancies occurred in seven women. In five women, after quinagolide withdrawal, the plasma PRL baseline values ranged from 52 to 158 micrograms/l and from 65 to 192 micrograms/l, respectively, at the first trimester and at the end of uneventful pregnancies. In contrast, in two women a rapid increase of PRL (240-400 micrograms/l) correlated with tumor growth during the first trimester. Such a tumor progression was blocked by quinagolide treatment but not by bromocriptine. These data, although observed in a limited series, justify the careful follow-up of pregnancies in this subclass of patients at risk. Finally, in the whole population, long-term control of hyperprolactinemia by quinagolide was obtained in 11/28 patients (39%) previously resistant to bromocriptine, and 15/20 women (75%) resumed normal gonadal function with a quinagolide daily dose of 300 micrograms in most of them.

Pit-1 gene expression in the human pituitary and pituitary adenomas.

The cellular basis for pituitary neoplasia is poorly understood. The POU domain protein Pit-1 is a pituitary-specific transcription factor involved in the generation, differentiation, and proliferation of three pituitary cell types: lactotrophs, somatotrophs, and thyrotrophs. In this study, we analyzed the expression of Pit-1 gene in a series of 15 different human pituitary tumors and compared it with that observed in normal tissue. Pit-1 transcripts, identical in size (2.4 and 4.5 kilobases) and sequence to those observed in normal tissue were evidenced in PRL-, GH-, and TSH-secreting tumors. Pit-1 is overexpressed (2.5- to 5-fold) in the PRL- and GH-secreting tumors, but to an extent consistent with the predominant cellular type of these adenomas. An isoform of Pit-1, with an insertion of 26 amino acids in the trans-activation domain as a result of alternative splicing, is also present in both normal and tumoral tissues. It is concluded that human pituitary tumorigenesis does not seem to be associated with a gross alteration of Pit-1 gene expression.

[Etiopathogenesis of pituitary tumors]. / Etiopathogénie des tumeurs hypophysaires.

In this present work, the authors discuss some recent advances in the pathogenesis of pituitary tumours. The model of transgenic mice suggest that chronic hormonal stimulation and some growth factors could sustain pituitary tumour development. However, these data are not suitable for human pituitary adenomas. The evidence that most pituitary adenomas are monoclonal in origin has prompted a search for somatic mutations. The mutated Gs alpha are found in only 30-40% of somatotroph adenomas and the ras mutations seem to be associated with the malignant transformation. In some prolactinomas resistant to the bromocriptine treatment, quantitative and qualitative alterations of the dopamine receptor D2, have been described. Mutations of protein kinase C have been identified in some invasive pituitary tumours. Molecular abnormalities have been reported in some cases (allele loss at the 11q13 locus, retinoblastoma gene mutation, aberrant expression of hst gene, Pit-1 overexpression) but none by itself can explain the tumour formation. The pituitary tumorigenesis is certainly a multistep process with the intervention of multiple promoting factors.

[Membrane receptors and coupling proteins in adenohypophyseal cells]. / Récepteurs membranaires et protéines de couplage dans les cellules adénohypophysaires.

Dopamine receptors of D2 type present on lactotroph cells are coupled to a large series of transduction mechanisms. Beside their negative coupling with adenylate cyclase, they are also coupled with potassium and calcium channels, leading to a decreased intracellular calcium concentration. In addition, D2 dopamine receptors also modulate phospholipase activities. Dopamine inhibits inositol phosphate production, through two distinct mechanisms. One of them could represent a direct negative coupling with phospholipase C. All these transduction mechanisms of the D2 dopamine receptors implicate G proteins sensitive to pertussis toxin. In contrast, these receptors are negatively coupled to phospholipase A2 through G proteins insensitive to this toxin. Both isoforms of the D2 dopamine receptor, generated by alternate splicing of a single gene are present in lactotroph cells. After transfection in CH4C1 cells the two isoforms are coupled with adenylate cyclase while only the shortest isoform appears negatively coupled to phospholipase C. Functional D2 dopamine receptors are present in human prolactinomas. Resistance to bromocriptine therapy is associated with a decrease density of these receptors in the tumor. In addition, the ratio of the two receptor isoforms (measured by PCR) is different in responsive and resistant tumors. Furthermore, the activity of Gi/Go proteins coupled to adenylate cyclase appears also affected in resistant tumors. Resistance to bromocriptine therapy appears thus to involve multiple changes at the different levels of the multiple mechanisms of action of dopamine on lactotroph cells.

The Nb2 form of prolactin receptor is able to activate a milk protein gene promoter.

We have recently cloned a cDNA encoding a mutant form of PRL receptor (PRL-R) from Nb2 cells, a PRL-dependent T lymphocyte-derived cell line. This cDNA is identical to the long form of the rat PRL-R, except for a deletion of 594 base pairs in the cytoplasmic domain, resulting in a mature receptor protein of 393 amino acids. Although a segment containing three cytoplasmic regions of moderate to high amino acid sequence identity with members of the PRL/GH receptor family is missing in this receptor form, the region of highest (70%) identity is retained. In the following studies, a homologous functional assay was developed to test the activity of three forms of receptor with respect to their ability to transmit a lactogenic signal. In this system, CHO cells were transiently transfected with a construct containing 2300 base pairs of the 5'-flanking sequence of the rat beta-casein gene fused to the chloramphenicol acetyltransferase (CAT) gene and an expression vector containing the various forms of rat PRL-R cDNA. The transfected cells were grown in serum-free medium in the absence or presence of PRL. In cells transfected with the long form of the PRL-R and beta-casein/CAT construct, a 7.2- +/- 0.9-fold induction (n = 3) of CAT activity was seen when cells were cultured in the presence of 400 ng/ml PRL and 1 micrograms/ml hydrocortisone. This level of stimulation was similar to that observed for the ovine beta-lactoglobulin/CAT construct in which a 5.7- +/- 1.2-fold (n = 3) effect was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of prolactin and its receptor in human lymphoid cells.

We have investigated whether human lymphoid cells are able to synthesize and secrete human PRL (hPRL) and to express PRL receptors. Metabolic labeling with [35S]methionine and immunoprecipitation of cell extracts from human mononuclear cells (MNC) and a human T lymphocyte cell line with an antiserum against hPRL revealed protein of M(r) 23,000, identical in size to pituitary hPRL. Dilution curves of lymphocyte immunoreactive hPRL were parallel to those obtained with pituitary hPRL in an immunoradiometric assay using two monoclonal antibodies against hPRL. Polymerase chain reaction experiments with primers located in the coding sequence of hPRL showed that the hPRL gene was expressed in MNC. Furthermore, cDNA cloning and sequence analysis indicated the presence of an extra 5' noncoding exon previously described for decidual hPRL. When MNCs were further separated into B cells, T cells, and monocytes, the expression of hPRL appeared to be mainly associated with the T lymphocyte fraction. The hPRL transcript was also detected in thymocytes and in a set of human lymphoid cell lines. Finally, polymerase chain reaction experiments revealed a ubiquitous distribution of PRL receptor gene expression in B cells, T cells, and monocytes. The presence of the receptor for PRL and production of PRL by T lymphocytes suggest a possible autocrine or paracrine effect of PRL in immune cell function.

Effects of the dopamine agonist CV 205-502 in human prolactinomas resistant to bromocriptine.

In 21 patients with prolactinomas resistant to bromocriptine, we studied the effects of CV 205-502 on PRL hypersecretion and tumor mass, as assessed by consecutive computed tomography examinations. Cell culture studies were performed in 9 of such tumors. In 11 patients (group I; 52%) with a mean baseline plasma PRL level of 468 +/- 160 micrograms/L (+/- SE), normal PRL values were achieved after 1-6 months of treatment with 0.1-0.5 mg/day CV 205-502. Tumor size was reduced by 25% or more in 6 of 11 patients. In group II (n = 10), PRL levels (948 +/- 538 micrograms/L at baseline) were reduced by 48% after treatment with 0.1 mg/day CV 205-502. A progressive increase in the daily dose up to 0.5 mg did not further improve the partial reduction of PRL. No reduction in tumor size was observed in this group. The cell culture studies showed that 1) a brief exposure to both drugs provoked PRL suppression lasting 3 days; 2) in group I, CV 205-502 suppressed PRL release more efficiently than bromocriptine, with a maximal inhibition of 72% at 10(-9) mol/L; and 3) in group II, CV 205-502 only achieved a 26% inhibition of PRL release at 10(-8) mol/L, superimposable to that of bromocriptine. These data indicate that in at least half of such adenomas resistant to bromocriptine, CV 205-502, probably due to its higher affinity toward the D2 dopamine receptor, can overcome such resistance.

D2 dopaminergic receptors: normal and abnormal transduction mechanisms.

Dopamine receptors of D2 type present on lactotroph cells are coupled to a large series of transduction mechanisms. Beside their negative coupling with adenylate cyclase, they are also coupled with potassium and calcium channels, leading to a decreased intracellular calcium concentration. In addition, D2 dopamine receptors also modulate phospholipase activities. Dopamine inhibits inositol phosphate production, through two distinct mechanisms. One of them could represent a direct negative coupling with phospholipase C. All these transduction mechanisms of the D2 dopamine receptors implicate G proteins sensitive to pertussis toxin. In contrast, these receptors are negatively coupled to phospholipase A2 through G proteins insensitive to this toxin. Both isoforms of the D2 dopamine receptor, generated by alternate splicing of a single gene, are present in lactotroph cells. After transfection in CH4C1 cells the two isoforms are coupled with adenylate cyclase while only the shortest isoform appears negatively coupled to phospholipase C. Functional D2 dopamine receptors are present in human prolactinomas. Resistance to bromocriptine therapy is associated with a decreased density of these receptors in the tumor. In addition, the ratio of the two receptor isoforms (measured by PCR) is different in responsive and resistant tumors. Furthermore, the activity of Gi/Go proteins coupled to adenylate cyclase appears also affected in resistant tumors. Resistance to bromocriptine therapy appears thus to involve multiple changes at the different levels of the multiple mechanisms of action of dopamine on lactotroph cells.

Prolactinomas and resistance to dopamine agonists.

Among 288 patients with prolactinoma (aged 12-62 years; 242 women), 27 were diagnosed as resistant to bromocriptine as their plasma prolactin (PRL) levels remained elevated despite long-term (3 months or more) treatment at high doses (> or = 15 mg daily). These 18 women and 9 men, aged 29 +/- 9 years (mean +/- SD, range 13-50), followed-up for 8 +/- 4 years, had microadenomas (n = 6) or macroadenomas. They were treated by dopamine agonists alone (n = 6) or associated with surgical or radiation therapy. In 8 cases repetitive surgical treatments were necessary. Among the 24 patients who were treated with the nonergot dopamine agonist CV 205-502 after unsuccessful bromocriptine treatment, half of them (9 women, 3 men) resumed normal PRL levels on doses ranging from 0.15 to 0.45 mg/day. Despite daily doses of CV 205-502 from 0.3 to 0.525 mg, the remaining patients were not normalized by this drug which did not prevent tumor growth in 4 of them. Two patients died from invasive cerebral extensions of their tumor and a third had vertebral metastases with positive anti-PRL immunostaining. It is concluded that bromocriptine-resistant prolactinomas represent the most severe aspect of this disease and that a more powerful dopamine agonist like CV 205-502 is effective in only a fraction of these patients.

A prolactin-dependent immune cell line (Nb2) expresses a mutant form of prolactin receptor.

The Nb2 cell line is a pre-T rat lymphoma that is dependent on prolactin (PRL) for mitogenesis. Two forms of PRL receptor (PRL-R), which differ in the length of their cytoplasmic domains have been identified in different tissues and species. In the present study we have cloned the cDNA and characterized the mitogenic form of PRL-R in Nb2 cells. Polymerase chain reaction amplification of first strand cDNA prepared from Nb2-11C (PRL-dependent) and Nb2-Sp (PRL-independent) cell lines was performed using oligonucleotide primers specific for the binding domain, the short form of the PRL-R, and the cytoplasmic domain of the long form of the PRL-R. These studies indicate that both cell lines express a novel form of PRL-R. A cDNA was isolated from an Nb2-Sp cDNA library, which contains 1446 base pairs identical to the nucleotide sequence of the long form of the rat PRL-R. However, the cDNA sequence is missing 594 base pairs in the cytoplasmic domain compared with the long form of the PRL-R. The cDNA encodes a protein of 393 amino acids, lacking 198 amino acids in the cytoplasmic domain. Scatchard analysis of 125I-labeled ovine prolactin (oPRL) binding to microsomes prepared from transiently transfected COS-7 cells with either PRL-R long form cDNA or Nb2 PRL-R cDNA indicates that the long form of PRL-R binds oPRL with high affinity (K alpha = 8.8 x 10(9) M-1), while the Nb2 PRL-R showed a 3.3-fold increased affinity for PRL (K alpha = 29.1 x 10(9) M-1). In addition, immunoblot analysis of these microsomes using 125I-labeled monoclonal antibody (U6) to the PRL-R demonstrates a Mr of approximately 82,000 for the long form and approximately 62,000 for the Nb2 form of PRL-R. Polymerase chain reaction amplification of genomic DNA prepared from PRL-dependent and -independent cell lines suggests that this form of PRL-R results from a deletion in the PRL-R gene. The identification of a modified long form of PRL-R in the Nb2 cell line should help localize domains of the PRL-R involved in signal transduction and further the investigation of prolactin's role in immune cell proliferation.

Different pathways of secretion for glycosylated and nonglycosylated human prolactin.

Human prolactinoma cells in culture secrete the monomeric nonglycosylated form of human PRL (NG-hPRL) and its glycosylated variant (G-hPRL). We have performed pulse-chase experiments to investigate the individual patterns of release of these two molecular variants. The cells were pulse labeled for 10 min with [35S]methionine and then chased for increasing periods of time up to 24 h. The secretion of newly synthesized G- and NG-hPRL was followed by immunoprecipitation of the chase medium and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Both forms were rapidly released (10 min of chase), but presented with different rates of secretion. Half-maximal release of G-hPRL occurred with 60-min chase, while 110 min were necessary for NG-hPRL. More than 50% of initially labeled G-hPRL was released in the medium vs. only 20% for NG-hPRL. Incubation of the cells with 8-chloroadénosine-cAMP during a 2-h chase period resulted in a 3.6-fold increase in the release of newly synthesized NG-hPRL and had only a slight effect on newly synthesized G-hPRL release (1.7-fold increase). The intracellular transit of labeled G- and NG-hPRL was investigated in cells treated by the ionophore monensin. The secretions of both newly synthesized forms were inhibited to the same extent, probably via an arrest of the transit at the level of the median Golgi, as judged by the delay of acquisition to endoglycosidase-H resistance for G-hPRL in monensin-treated cells. In contrast, Western blot analysis of the same medium-showed that monensin abolished the secretion of G-hPRL and had little effect on NG-hPRL. Our results on the different rates of secretion of G- and NG-hPRL indicate a sorting of the two forms into different compartments in the secretory pathway, with G-hPRL being secreted at a higher rate than NG-hPRL, possibly via a different intracellular route. The differential effects of 8Cl-cAMP and monensin further suggest that G-hPRL may be constitutively secreted after synthesis, while NG-hPRL secretion may involve a storage step.