help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ham, J.
Right arrow Articles by Scanlon, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ham, J.
Right arrow Articles by Scanlon, M. F.
The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 5 1598-1603
Copyright © 1998 by The Endocrine Society

Original Studies

Immortalized Human Pituitary Cells Express Glycoprotein {alpha}-Subunit and Thyrotropin ß (TSHß)

J. Ham, J. Webster, J. A. Bond, B. Jasani, M. D. Lewis, P. J. Hepburn, J. S. Davies, B. M. Lewis, D. Wynford Thomas and M. F. Scanlon

Departments of Medicine (J.H., J.W., M.D.L., P.J.H., J.S.D., B.M.L., M.F.S.), and Pathology (J.A.B., B.J., D.W.T.), University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, Wales, United Kingdom

Address all correspondence and requests for reprints to: Dr. J. Ham, Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, Wales, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A major problem in the study of human pituitary cells is their lack of proliferative capacity in vitro. To address this issue, we have infected normal human, postmortem pituitary cells in monolayer culture with a temperature-sensitive (tsA58) mutant of SV40 large T antigen. Several epithelial-like colonies were isolated; and one, designated CHP2, has been studied in detail to identify its functional characteristics. CHP2 cells have undergone more than 150 culture passages and retain an epithelial morphology. They exhibit tight temperature-dependent growth, in the presence and absence of serum, with cell division at 33 C and growth inhibition at 39 C. CHP2 cells, at both temperatures, showed diffuse immunostaining for human {alpha}-subunit and focal staining for TSHß. Gene expression was confirmed by RT-PCR and sequencing. TRH and GnRH receptors were not detectable, and their absence was confirmed by their lack of effects on intracellular calcium and inositol phospholipids. Cytogenetic analysis showed that the cells had a modal peak in the diploid range and a smaller peak in the tetraploid range. There was also a consistent loss of chromosome 22 and a normal chromosome 2 homologue, the latter being replaced by one of two chromosome 2 markers, M2A or M2B. In conclusion, we have immortalized human pituitary cells using SV40 tsT, from which we have cloned a cell line expressing {alpha}-subunit and TSHß.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EXISTENCE of cell lines in which specific differentiated phenotypes are preserved has greatly facilitated the study of cellular and molecular biology by providing homogeneous populations of well-characterized cells. Our understanding of the endocrinology and molecular biology of the anterior pituitary hormones, GH, PRL, and POMC, has been greatly advanced by the availability of the cell lines GH3 (1) of rat somatotrophic-lactotrophic origin, and AtT-20 derived from mouse corticotrophs (2). Other workers have described the apparently spontaneous development of human pituitary GH-secreting and PRL-secreting cell lines, but these were of limited lifespan (3, 4) or underwent spontaneous involution (5). Chomczynski et al. (6) recently reported the development of the GX human pituitary cell line, which possesses GH messenger RNA (mRNA) but has lost the ability to secrete GH.

The lack of analogous human cell lines of glycoprotein-secreting lineage has made study of thyrotroph and gonadotroph function more difficult; primary cultures have limited viability and progressively lose differentiated function (7). Targeted oncogenesis has been used to develop mouse cell lines of glycoprotein-secreting lineage (8, 9). Transgenic mice were developed with SV40 large T antigen (Tag) expression driven by the 5' flanking region of the human glycoprotein hormone {alpha}-subunit gene. Large pituitary tumors did indeed develop, but the resulting cell lines expressed only {alpha}-subunit, and none of the ß-subunits (which provide the specificity of action of the {alpha}ß heterodimer). This same group (10) subsequently used a larger (1.8-kb) 5' flanking region and identified mouse pituitary cells expressing both TSHß and {alpha} subunits. Similar technology, using the rat LHß subunit regulatory region, resulted in cells expressing both {alpha}- and ß-subunits of LH and the GnRH receptor (10).

An approach that has been used successfully with human thyroid epithelium involves the use of a mutant gene encoding a temperature-sensitive variant of the early region Tag of SV40 (tsT) (11). This gene, carried into cells in an amphotropic retroviral vector, is capable of transforming cells at the permissive temperature (33 C), but its Tag product is inactivated at the restrictive temperature (39–41 C), offering the potential for reversible immortalization of the cells. We have used this procedure, in an attempt to immortalize human pituitary cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary human pituitary cultures

Human anterior pituitary tissue was removed 3 h postmortem from a female patient, 69 yr old, who died after cardiac surgery. The tissue was washed in Earle’s balanced salt solution and dispersed with collagenase (1 mg/mL), hyaluronidase (1 mg/mL) and deoxyribonuclease I (0.25 mg/mL) at 37 C, with intermittent mechanical trituration. Cells were plated out at 5 x 105 cells/60-mm petri dish in DMEM and 10% FCS, as previously described (12).

Production of retroviral ts.SV40T vector and retroviral infection

The retroviral ts.SV40T vector was prepared by infection of the amphotropic packaging line {psi}-CRIP with supernatant from the ecotropic producer line {psi}-2-ts A58 U19 (13) (kindly donated by Dr. M. O’Hare, ICR, Sutton, UK). Supernatant from the producer cells {psi}-CRIP-ts A58 U19 clone 2 (11) was applied to the pituitary cell cultures for 2 h at 33 C in the presence of polybrene (8 µg/mL). Two days were allowed for proviral integration and infection. Cells were then subsequently maintained at 33 C in MCDB 104 (25%), DMEM (50%), and Ham’s F12 (25%), containing FCS (10%) and G418 (400 µg/mL). Cultures were then inspected for foci of developing cells; these were subsequently isolated, using cloning rings, and detached with 0.05% trypsin and subcultured at 33 C in the media described above. G418 was used to select for the presence of the retroviral vector and was omitted from the growth media after 20 culture passages.

Cytogenetic chromosome analysis

Cells for cytogenetic analysis were processed using standard procedures, and the trypsin/Leishman combination was used for chromosome staining and banding. Ten metaphase spreads were analyzed and karyotyped using an A11 Cytoscan3 computer (Applied Imaging Ltd. Sunderland, UK).

Growth characteristics

CHP2 cells (3 x 104) were plated into 35-mm petri dishes in the presence of 10% FCS and incubated at 33 C and 39 C. The culture media were changed at 3-day intervals. The effect of serum was tested by incubating cells at 33 C in the absence of FCS and at concentrations of 2% and 10% FCS. Cells in replicate cultures were counted in a Coulter counter.

Immunocytochemistry

Immunostaining for SV40 large T protein and cytokeratins was carried out as previously described (13). Antisera to anterior pituitary hormones were obtained from NIAMDD (Bethesda, MD), and the anticytokeratins AE1/AE3 and CAM5.2 were obtained from Dako Ltd. (High Wycombe, UK) and Becton Dickinson Ltd. (Oxford, UK), respectively. All antisera were used at the suggested concentrations and were visualized using an indirect immunoperoxidase procedure.

RT-PCR

{alpha}-Subunit and TSHß RT-PCR was performed in a single step using 1 µg total RNA, prepared using Trizol reagent (Life Technologies, Paisley, UK). The PCR forward and reverse primer sequences corresponded to bp 72–98 and bp 348–377 for {alpha}-subunit (14) and 294–316 and 1058–1087 for TSHß (15), yielding, respectively, 306-bp and 345-bp fragments. The PCR products were analyzed on an ABI Prism 377 sequencer (Perkin Elmer, Warrington, UK). The primers for the human TRH receptor corresponded to bp 330–354 and bp 784–807 (16), and those for the GnRH receptor were as described elsewhere (17).

Inositol phospholipid turnover

CHP2 cells were plated into 24-well multidishes at a density of 2 x 104/cm2. After 4 days in culture at 33 C, cells were labeled for 3 days with [3H]-myoinositol (80–120 Ci/mmol, 0.5 µCi/0.5 mL/well) in inositol-free, serum-free DMEM. Cultures were treated with agonists in the presence of 10 mmol/L lithium chloride, as described in the text. Inositol monophosphate was extracted and quantified as previously described (18).

Cytosolic Ca2+

Cytosolic free Ca2+ changes were measured using ratiometric fluorometry of fura-2 loaded cells, as previously described (19).

cAMP measurements

cAMP was measured by specific RIA, as previously described (20).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of epithelial cells after retrovirus vector tsT infection

Six weeks after tsT infection of the normal human pituitary cells, six foci of mitotically active cells with an epithelial morphology were observed within a fibroblastoid monolayer. These colonies were designated CHP1–6, two of which (CHP2 and CHP3) have been isolated and passaged through, respectively, >150 and >75 population doublings. The CHP2 cells have now been extensively studied. At the permissive temperature of 33 C, they showed an epithelial-like morphology with large nuclei and abundant cytoplasm (Fig. 1aGo). At 39 C, however, more of the cells showed a fibroblastic appearance (Fig. 1bGo). Electron microscopy revealed well-developed Golgi apparatus and abundant mitochondria but few secretory granules. More than 90% of the cells at 33 C expressed SV40 Tag (data not shown), as determined by immunostaining with PAb419 monoclonal antibody (13).



View larger version (129K):
[in this window]
[in a new window]
  		Figure 1. Phase contrast micrographs of CHP2 cells in monolayer culture (a) at 33 C and (b) after 72 h at 39 C (x 200).

 
Chromosome analysis

Chromosome counts indicated a modal peak (44 chromosomes) in the diploid range and a smaller peak (72–82 chromosomes) in the tetraploid range (Fig. 2Go). The exact number of chromosomes per cell was also variable. In the ten cells examined, the only consistent chromosomal change was the loss of chromosome 22. All cells had also lost a normal chromosome 2 homologue and had gained one of two marker chromosome 2s (M2A or M2B) (Table 1Go). Both markers seemed to have deletions of the short arm of chromosome 2. Other chromosomal losses included 7 and 10; these were nonrandom but were not seen in all cells. Chromosome gain was seen in only three cells and was random. Other unidentified single-cell markers, typical of SV-40 cells, were also observed.



View larger version (9K):
[in this window]
[in a new window]
  		Figure 2. Chromosome counts in CHP-2 cells vs. cell number (44 cells were counted).

 

View this table:
[in this window]
[in a new window]
  		Table 1. Chromosomal changes in CHP-2 cells

 
Growth characteristics

Growth studies showed that the population-doubling time for CHP2 at 33 C was about 3 days. At 39 C, the cell number remained unchanged over a period of 7 days but fell to about 60% of the initial number plated out at 10 days (Fig. 3Go). At the permissive temperature, cell division occurred, in the absence of serum, with around 20% increase in cell number after 4 days. Over the same time scale, the presence of 2% serum caused a doubling in the number of cells, but raising the serum concentration to 10% caused no further increase (Fig. 3Go).



View larger version (9K):
[in this window]
[in a new window]
  		Figure 3. Growth of CHP2 cells at 33 C ({blacktriangleup}) and 39 C (•) in 10% FCS (top panel) and in different FCS concentrations (•, 0%; {blacktriangleup}, 2%; {blacksquare}, 10%) (bottom panel). Values are means ± SEM (n = 4).

 
Immunocytochemistry of cytokeratins and anterior pituitary hormones

Immunocytochemistry was performed on cells incubated at 33 C and for up to 7 days at 39 C. Antiserum AE1/AE3 (a broad-spectrum cytokeratin antisera that also recognizes keratinizing and nonkeratinizing squamous epithelia) showed strong positive staining in many of the cells, whereas antiserum CAM5.2, which recognizes simple and glandular epithelia, showed only focal staining (Fig 4Go). Staining with both antisera was markedly increased at 39 C. Diffuse cytoplasmic staining to the common {alpha}-subunit was observed at 33 C, whereas only focal staining was seen at 39 C. The difference in the pattern of {alpha}-subunit staining at the two temperatures was in contrast to that seen for the cytokeratins. A few cells were labeled with anti-TSHß (Fig. 5Go) at both temperatures. None of the other glycoprotein hormones were detectable. There was no evidence of GH, PRL, or ACTH at either temperature, and there was no reaction with the folliculo-stellate marker S-100 or chromogranin A antibodies. Neuron-specific enolase, however, was detectable in a few cells.



View larger version (146K):
[in this window]
[in a new window]
  		Figure 4. Immunocytochemical labeling of cytokeratins with antisera CAM 5.2 (a and b) and AE1/AE3 (c and d) in CHP2 cells, incubated at 33 C (a and c) and 39 C (b and d).

 


View larger version (145K):
[in this window]
[in a new window]
  		Figure 5. Immunocytochemical labeling of {alpha}-subunit (a and b) and TSHß (c and d) in CHP-2 cells incubated at 33 C (a and c) and 39 C (b and d). (The arrows indicate TSHß positive cells).

 
RT-PCR

Major bands of the expected size (306 bp for {alpha}-subunit and 345 bp for TSHß) were amplified from RNA of CHP2 cells cultured at both 33 C and 39 C. The sequences corresponded to those published (14, 15). TRH and GnRH receptor mRNAs were not detectable.

Inositol phospholipid, cAMP, and Ca2+ responses

TRH and GnRH had no effect on cAMP, inositol phosphates (IP), or Ca2+ levels in CHP2 cells at both 33 C and 39 C. Forskolin (10 µmol/L; 15 min exposure) stimulated intracellular cAMP levels at both temperatures (169 ± 7% and 177 ± 17% of basal, respectively; P < 0.01). Carbachol (0.1 mmol/L), on the other hand, stimulated a doubling of IP and Ca2+ at both temperatures (Table 2Go). CRH, GHRH, vasoactive intestinal peptide, and pituitary adenylate cyclase activating polypeptide, at concentrations up to 1 µmol/L, had no effect on cAMP levels.


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of carbachol (0.1 mmol/L) on IP and free Ca2+ levels in CHP-2 cells cultured at 33 C and 39 C (72 h)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we describe the immortalization of human anterior pituitary cells, obtained at postmortem, using a temperature-sensitive mutant of SV40 Tag. Out of six transformed colonies initially identified, one has been extensively studied. These cells were shown to be essentially diploid, with a human chromosome complement. The cells have been maintained in culture for over 2 yr and have undergone in excess of 150 culture passages. The tsT has been used successfully to immortalize a variety of tissues, including fibroblasts (21), keratinocytes (22), central nervous system (23), liver (24), and adult human thyroid (13). The theoretical advantage of using tsT is to allow study of the transformed cells at the restrictive temperature, in the presence of an inactive configuration of T. In several cases, incubating the cells at the restrictive temperature led to loss of the transforming phenotype concomitant, with increased expression of several different antigens. For example, prolonged incubation of tsT-transformed liver cells, at 40 C, led to increased synthesis of {alpha}-fetoprotein, albumin, and transferrin (24); and similarly, the expression of keratins and cross-linked envelopes (22) in tsT epidermal cells was increased at the restrictive temperature.

In our experiments, cytokeratin expression was also increased at the restrictive temperature, whereas that for {alpha}-subunit and TSHß was, respectively, reduced and unchanged. The use of RT-PCR and sequencing confirmed the synthesis of {alpha}-subunit and TSHß. None of the other pituitary hormones were detectable by immunocytochemistry or by RT-PCR. The message for the transcription factor pit-1 (which is normally present in GH, PRL, and TSH producing cells) was absent. The reason for this is unclear; however, the absence of pit-1 and TRH and GnRH receptors in these cells may indicate a primitive state of differentiation and a loss of normal physiological control mechanisms.

Karyotype analysis of CHP2 cells showed a chromosomal count typical of SV-40 transformed cells in culture. The consistent loss of chromosome 22 and the presence of two chromosome 2 markers (M2A and M2B) in nine of ten cells examined suggests that the cells are probably derived from a single transformed cell. Although it is clear that CHP2 cells express {alpha}-subunit and TSHß mRNA, and synthesize the corresponding peptides, we were unable to demonstrate their secretion. The lack of secretory granules, on electron microscopy analysis, may indicate that the cells of origin are a type of null cell (which may explain the lack of detectable secretion). Some null cells, however, do contain glycoprotein hormone subunits, the most common being {alpha}-subunit (although GH, PRL, and POMC can sometimes be present too) (25).

The adenylate cyclase/cAMP system was active in these cells, but there was no evidence of linkage to CRH, GHRH, vasoactive intestinal peptide, and pituitary adenylate cyclase activating polypeptide receptors. As expected, TRH and GnRH, due to the lack of their respective receptors, had no effect on IP and Ca2+ accumulation. On the other hand, carbachol (but not adrenaline) did stimulate IP and Ca2+ at both the permissive and restrictive temperatures. Clearly, the muscarinic M1 receptor remains functional, although its role in pituitary growth or function is hitherto unknown.

In conclusion, we have immortalized human pituitary cells with tsT and one of the clones, CHP2, has been maintained in continuous culture for over 2 yr. The cells express {alpha}-subunit and TSHß. Whether the expression of {alpha}-subunit and TSHß are under any form of regulatory control is currently under investigation. The cells proliferate rapidly at 33 C but cease growth at 39 C, which may facilitate the study of hormone regulation in the presence of inactive T. These cells can also be used to incorporate activated oncogenes, and their mechanisms of action can subsequently be studied at the restrictive temperature. Such a system could thus provide a model for studying human epithelial endocrine cell neoplasia.


   Acknowledgments
 
We are grateful to Dr. M. Hallett (Department of Surgery) for performing the Ca2+ studies; to NIAMDD and NHPP, University of Maryland School of Medicine, for the supply of peptide antisera; and to Istituto di Ricerca, Cesare Serono, Roma, Italy, for their generous support. We also would like to thank Dr. J. J. Waters, Regional Cytogenetics Laboratory, Birmingham, for the cytogenetic analysis studies.

Received July 30, 1997.

Revised January 23, 1998.

Accepted January 27, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tashjian AM, Yamura Y, Levine L, Sato GH, Parker ML. 1988 Establishment of clonal strains of rat pituitary cells that secrete growth hormone. Endocrinology. 82:342–352.[Medline]
  2. Buonassisi V, Sato G, Cohen AI. 1962 Hormone producing cultures of adrenal and pituitary origin. Proc Natl Acad Sci USA. 48:1184–1190.[Free Full Text]
  3. Dufy-Barbe L, Corcuff JB, Sartor P, Guerin J, Dufy B. 1989 Human pituitary cell lines derived from somatomammotropic adenoma: response to growth hormone-releasing hormone (GHRH). J Endocrinol Invest. [suppl 2] 12 (Abstract 12).
  4. Dufy-Barbe L, Corcuff JB, Sartor P, Guerin J, Dufy B. Neuropeptide expression in human pituitary cell lines. Proc of the 2nd International Pituitary Congress, Palm Springs, CA, 1989, (Abstract T-27).
  5. Prysor-Jones RA, Silverlight JJ, Jenkins JS. 1989 Oestradiol, vasoactive intestinal peptide and fibroblast growth factor in the growth of human pituitary tumour cells in vitro. J Endocrinol. 120:171–177.[Abstract]
  6. Chomczynski P, Sosynski PA, Frohman LA. 1993 Stimulatory effect of thyroid hormone on growth hormone gene expression in a human pituitary cell line. J Clin Endocrinol Metab. 77:281–285.[Abstract]
  7. Tougard C, Tixier-Vidal A. 1988 Lactotrophs and gonadotrophs. In: Knobil E, Neill JD, eds. Physiology of reproduction, ch. 30. New York: Raven Press; 1305–1333.
  8. Windle J, Weiner R, Mellon P. 1990 Cell lines of pituitary gonadotroph lineage derived by targeted oncogenesis in transgenic mice. Mol Endocrinol. 4:597–603.[Abstract]
  9. Mellon PL, Windle JJ, Weiner RI. 1991 Immortalisation of neuroendocrine cells by targeted oncogenesis. Recent Prog Horm Res. 47:69–96.
  10. Schechter J, Windle JJ, Stauber C, Mellon PL. 1992 Neural tissue within anterior pituitary tumours generated by oncogene expression in transgenic mice. Neuroendocrinology. 56:300–311.[Medline]
  11. Wynford-Thomas D, Bond JA, Wyllie FS, et al. 1990 Conditional immortalisation of human thyroid epithelial cells: a tool for the analysis of oncogene action. Mol Cell Biol. 10:5365–5377.[Abstract/Free Full Text]
  12. Webster J, Ham J, Bevan JS, ten Horn CD, Scanlon MF. 1991 Preliminary characterisation of growth factors secreted by human pituitary cells. J Clin Endocrinol Metab. 72:687–692.[Abstract]
  13. Wyllie FS, Bond JA, Dawson T, White D, Davis R, Wynford-Thomas DW. 1992 A phenotypically and karyotypically stable human thyroid epithelial line conditionally immortalised by SV40 large T antigen. Cancer Res. 52:2938–2945.[Abstract/Free Full Text]
  14. Fiddes JC, Goodman HM. 1979 Isolation, cloning and sequence analysis of the cDNA for the {alpha}-subunit of human chorionic gonadotropin. Nature. 281:351–356.[CrossRef][Medline]
  15. Tatsumi K, Hayashizaki Y, Hiraoka Y, Miyai K, Matsubara K. 1988 The structure of the human thyrotropin beta-subunit gene. Gene. 73:489–497.[CrossRef][Medline]
  16. Matre V, Karlsen HE, Wright MS, et al. 1993 Molecular cloning of a functional human thyrotropin-releasing hormone receptor. Biochem Biophys Res Commun. 195:179–185.[CrossRef][Medline]
  17. Sanno N, Jin L, Qian X, et al. 1997 Gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor messenger ribonucleic acids expression in nontumorous and neoplastic pituitaries. J Clin Endocrinol Metab. 82:1974–1982.[Abstract/Free Full Text]
  18. Ham J, Scanlon MF. 1994 The glutamate inhibition of carbachol stimulated inositol phosphate production in rat cortical cells is mediated through an ionotropic NMDA receptor. Neurosci Lett. 167:63–66.[CrossRef][Medline]
  19. Al-Mohonna FA, Hallett MB. 1988 The use of fura-2 to determine the relationship between cytoplasmic free calcium and oxidase activation in rat neutrophils. Cell Calcium. 9:17–26.[CrossRef][Medline]
  20. Lotwick HS, Haynes LW, Ham J. 1990 Glycyl-L-glutamine stimulates the accumulation of A12 acetylcholinesterase but not of nicotinic acetylcholine receptors in quail embryonic myotubes in a cAMP independent mechanism. J Neurochem. 54:1122–1129.[CrossRef][Medline]
  21. Jat PS, Sharpe PA. 1989 Cell lines established by a temperature-sensitive simian virus 40 large T antigen are growth restricted at the non-permissive temperature. Mol Cell Biol. 9:1672–1681.[Abstract/Free Full Text]
  22. Banks-Schlegel SP, Howley PM. 1983 Differentiation of human epidermal cells transformed by SV-40. J Biol Cell. 96:330–337.[Abstract/Free Full Text]
  23. Frederiksen K, Jat PS, Valtz N, Levy D, McKay R. 1988 Immortalisation of precursor cells from the mammalian CNS. Neuron. 1:439–448.[CrossRef][Medline]
  24. Chou JY, Schlegel-Haueter SE. 1981 Study of liver differentiation in vitro. J Cell Biol. 89:216–222.[Abstract/Free Full Text]
  25. Asa SL, Kovacs K, Melmed S. 1995 The hypothalamic-pituitary axis. In: Melmed S, ed. The pituitary. Cambridge: Blackwell Science Inc: 3–44.



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
D. C. Danila, X. Zhang, Y. Zhou, G. R. Dickersin, J. A. Fletcher, E. T. Hedley-Whyte, M. K. Selig, S. R. Johnson, and A. Klibanski
A Human Pituitary Tumor-Derived Folliculostellate Cell Line
J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 1180 - 1187.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ham, J.
Right arrow Articles by Scanlon, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ham, J.
Right arrow Articles by Scanlon, M. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals