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A R Buckley

Publications and source records attributed to A R Buckley.

At least 37 records · Page 2Linked to original sources
PubMed1999

Proteolysis of heat shock transcription factor is associated with apoptosis in rat Nb2 lymphoma cells.

Previously, we reported that prolactin (PRL)-dependent Nb2 lymphoma cells exhibit an aberrant heat shock response because of cysteine protease-mediated fragmentation of the heat shock transcription factor (HSF). Moreover, exposure of the cells to PRL abrogated heat-induced HSF proteolysis. The present study was conducted to investigate whether HSF proteolysis is a component of the apoptotic process in this model. Initially, the effect of heat stress (41 degrees C for 1 h) on apoptosis, determined by agarose gel electrophoresis and flow cytometric analysis, was evaluated in PRL-dependent Nb2-11 cells and in an autonomous subline (Nb2-SFJCD1). Heat was found to induce HSF proteolysis concomitant with activation of apoptosis in each cell line; treatment with PRL blocked these effects. To determine whether HSF proteolysis occurred as a generalized phenomenon associated with apoptosis, the effects of other activators of this process were evaluated. Vinblastine, cycloheximide, and thapsigargin stimulated fragmentation of HSF and hydrolysis of DNA in each cell line. The addition of PRL blocked the effects of vinblastine but was ineffective in cells treated with either cycloheximide or thapsigargin. Iodoacetamide, a cysteine protease inhibitor that blocks HSF fragmentation, also inhibited apoptosis. In addition, Z-VAD, a general caspase antagonist, blocked vinblastine-induced fragmentation of HSF and DNA, suggesting that the enzyme responsible for proteolysis of the transcription factor was likely a caspase family member. The results suggest that proteolysis of HSF reflects the action of one or more caspases activated as a consequence of stimulation of cell death. It is concluded that HSF may represent a previously unrecognized substrate for caspases or other cysteine proteases activated during apoptosis.

Alkylating Agents↗
PubMed1998

Changes in glutathione redox cycling and oxidative stress response in the malignant progression of NB2 lymphoma cells.

Differential analysis of closely related Nb2-lymphoma cell lines can be used for identification of changes in biochemical properties associated with the malignant progression of certain T-cell cancers. As tumors progress, they tend to show metabolic alterations such as an increased resistance to oxidative stress, a characteristic that may be correlated with changes in intrinsic antioxidant levels (e.g., glutathione) and in activities of associated enzymes such as the glutathione redox pathway. Whether increases in malignancy of Nb2 cells were associated with changes in cellular glutathione levels and activities of glutathione-metabolizing enzymes was addressed. To evaluate this relationship, 3 cell lines, showing increased malignancy, were used: Nb2-U17 (hormone-dependent, non-metastatic), Nb2-11 (hormone-dependent, metastatic), Nb2-SFJCD1 (growth factor-independent, metastatic). Compared to Nb2-U17 and Nb2-11 cells, the highly progressed Nb2-SFJCD1 lymphoma cells maintain low basal glutathione levels. However, the Nb2-SFJCD1 cells display an enhanced capacity to produce glutathione when challenged with an oxidative stress and show a significantly higher resistance to H2O2-induced apoptosis.

Animals↗
PubMed1998

Prolactin stimulates phosphorylation of the human T-cell antigen receptor complex and ZAP-70 tyrosine kinase: a potential mechanism for its immunomodulation.

Prolactin (PRL) is an immunomodulatory hormone which promotes T-cell activation and proliferation. However, the intracellular mechanisms of this action in normal lymphocytes are unknown. Because the PRL receptor (PRLR) activates several signals also activated by the T-cell antigen receptor (TCR)/CD3 complex, we evaluated whether signaling "cross-talk" occurs between these distinct receptors. Using human thymocytes, human peripheral blood lymphocytes and the rat Nb2 lymphoma T-cell, we found that PRL induced rapid phosphorylation of multiple, TCR/CD3 complex proteins, an event required for lymphocyte activation. Two of these phosphorylated proteins were identified to be CD3 epsilon and ZAP-70 tyrosine kinase, molecules essential for TCR function. Further, PRL induced tyrosyl phosphorylation of ZAP-70 in each population of T-lymphocytes tested, demonstrating for the first time that ZAP-70 is a target of PRL action. Taken together, our results suggest that the PRLR directly affects T-lymphocyte activation by means of signaling cross-talk with the TCR/CD3 complex.

Adjuvants, Immunologic↗
PubMed1998

Heat-induced proteolysis of HSF causes premature deactivation of the heat shock response in Nb2 lymphoma cells.

Nb2-11 cells, a prolactin (PRL)-dependent T-lymphoma cell line, display an unusual response to heat stress characterized by the lack of expression of inducible hsp70 mRNA transcripts and a reduction in the levels of constitutively expressed heat shock protein (HSP) genes. This aberrant heat shock response appears to result from heat-induced proteolytic fragmentation of heat shock factor (HSF). In this report, we have investigated processes that promote HSF fragmentation and identified characteristics of a protease that may be responsible for this effect. Cycloheximide did not affect HSF fragmentation of heat-shocked Nb2-11 cells suggesting that proteases responsible for this proteolysis are constitutively expressed and become activated by the heat shock conditions. PRL protected Nb2-11 cells from heat-induced fragmentation whereas sodium butyrate (NaBT) rendered a fragmentation-resistant cell line (Nb2-SFJCD1 cells) sensitive to HSF proteolysis. Heat-induced HSF fragmentation in Nb2-11 cells was not affected by pretreating cultures with several serine protease inhibitors. However, a dose-dependent decrease in HSF fragmentation was achieved by pretreating cultures with iodoacetamide, a cysteine protease inhibitor that is active in apoptosis. Apparently, the heat shock response in Nb2 cells is attenuated by a mechanism that involves the premature deactivation of HSF by its selective proteolysis. Attenuation of this critical cellular stress response may be an important contributor to the progression of hormone-dependent tumors possibly by influencing apoptotic processes known to regulate the activity of these cells.

Animals↗
PubMed1998

Prolactin-regulated apoptosis of Nb2 lymphoma cells: pim-1, bcl-2, and bax expression.

Lactogen-dependent Nb2 lymphoma cells, widely employed for studying prolactin (PRL) mitogenic mechanisms, are also useful for investigations of apoptosis in T-lineage lymphocytes. Utilizing PRL-dependent Nb2-11 cultures, apoptosis-regulatory genes were evaluated for participation in dexamethasone- (DEX) provoked cell death or its inhibition by PRL. Treatment of lactogen-starved, G1-arrested Nb2-11 cells with DEX (100 nM) activated apoptosis within 12 h evaluated by flow cytometric analysis of fragmented DNA. This effect was not associated with altered expression of bcl-2, bax, or pim-1. PRL (10 ng/mL), coincubated with DEX-treated cells, completely blocked DEX-induced apoptosis. This inhibition was associated with increased expression of bcl-2 and pim-1 mRNAs, genes reported to suppress apoptosis, within 2-6 h after addition of the hormone. Moreover, the increased transcription of bcl-2 and pim-1 was coupled to increases in their protein levels. The results suggest that bcl-2, bax, and pim-1 do not play a critical role in DEX-induced apoptosis in Nb2 cells. However, expression of bcl-2, together with pim-1, may have a role in mediating the antiapoptotic actions of PRL.

Animals↗
PubMed1998

Prolactin receptor signaling: shared components with the T-cell antigen receptor in Nb2 lymphoma cells.

Previously, we reported that activation of the human prolactin receptor (PRLR) produced a protein phosphorylation pattern strikingly similar to that provoked by Concanavalin A (Con A), an activator of the T-cell antigen receptor (TCR). These results suggested that certain signaling components of the TCR may be shared by the activated PRLR. Additional studies here assessed the levels of TCR expression following PRLR stimulation and the effect of TCR activation on PRL-stimulated proliferation in lactogen-dependent pre-T Nb2-11 lymphoma cells. The results indicated that the TCR was expressed on the surface of approx 4% of exponentially proliferating and prolactin- (PRL) treated cells. In contrast, approx 45% of quiescent cells, cultured in the absence of PRL for 24 h, expressed the TCR at the cell surface, suggesting that lactogen withdrawal may up-regulate TCR cell-surface expression. Moreover, TCR activation with anti-CD3 antibodies attenuated PRL-stimulated Nb2-11 cell proliferation in a concentration-dependent manner. In other experiments, immunoprecipitation and immunoblotting of Nb2-11 lysates revealed that activation of the PRLR resulted in rapid tyrosyl phosphorylation of ZAP-70, a critical TCR-associated tyrosine kinase. In addition, ZAP-70 was found to associate transiently with the putative guanine nucleotide exchange factor and substrate, Vav, in PRL-treated cells. ZAP-70 was also found to associate constitutively with the PRLR; PRL stimulation provoked the transient recruitment of Vav to the complex. These observations suggest that PRL signaling reflects the transient formation of a PRLR-ZAP-70-Vav complex and its immunomodulatory actions involve diverse interactions that affect TCR expression and signaling mechanisms.

CD3 Complex↗
PubMed1997

Butyrate-induced reversal of dexamethasone resistance in autonomous rat Nb2 lymphoma cells.

The parental rat Nb2 lymphoma is a prolactin (PRL)-dependent T cell line. Exposure of a PRL-independent subline, Nb2-SFJCD1, to sodium butyrate (NaBT) causes transient reversal of their growth factor-independent proliferation in association with constitutive expression of protooncogenes pim-1 and c-myc. In the present study, we investigated the effect of NaBT treatment on the sensitivity of Nb2-SFJCD1 cells to dexamethasone (DEX)-induced apoptosis. Pretreatment with NaBT (2 mM, 72 h) partially reversed resistance to apoptosis in Nb2-SFJCD1 cells exposed to DEX (100 nM) for 12 h, assessed by flow cytometric analyses of DNA fragmentation. However, the cytolytic effect of DEX was abrogated by PRL in a time- and concentration-dependent manner. Evaluation of apoptosis-associated gene expression in NaBT-pre-treated cultures incubated with DEX or DEX+PRL indicated that the apoptosis resistance did not stem from altered bcl-2 or bax expression. However, there was a strong correlation between the resistance to DEX-activated apoptosis and their enhanced expression of pim-1 mRNA and protein. The results show that it is possible to reverse DEX-induced apoptosis of Nb2 pre-T cells and suggest the pim-1 gene product has an important role as a suppressor of this process, perhaps functioning as a mediator of PRL action.

Journal Article↗
PubMed1997

Increased cystine uptake capability associated with malignant progression of Nb2 lymphoma cells.

Analysis of rat, pre-T cell 'Nb2 lymphoma' sublines, manifesting different degrees of malignant progression, can indicate phenotypic changes potentially useful as therapeutic targets. In this study, the prolactin (cytokine)-dependent Nb2-11 and autonomous Nb2-SFJCD1 sublines were compared for in vitro thiol growth requirements. Whereas Nb2-11 culture growth depended on 2-mercaptoethanol (2-ME; 33-100 microM), Nb2-SFJCD1 cells were 2-ME-independent. This difference stemmed from differential uptake of exogenous L-cystine, critically required for proliferation. Uptake of 35S-L-cystine (10 microCi/ml; 40 microM) showed Nb2-11 cells had low cystine uptake capability; 2-ME enhanced cystine uptake to growth-sustaining levels. Nb2-SFJCD1 cells did not require 2-ME due to intrinsic, 11-fold higher cystine uptake via the x(c)- cystine/glutamate transport system. In absence of 2-ME, monosodium glutamate abrogated Nb2-SFJCD1 proliferation by specifically inhibiting cystine uptake (85% at 10 mM). Elevated glutathione (GSH) levels were not essential for growth of either line as shown with L-buthionine-(S,R)-sulfoximine (0.1-4 mM) treatment. The cyst(e)ine requirement therefore did not primarily involve maintenance of normal GSH levels, reported critical for T lymphocyte replication. These and other results suggest increased cystine uptake capability constitutes another potential step in progression of T cell cancers which is not coupled to cytokine autonomy or metastatic ability development. The x(c)- transport system apparently provides a novel target for T cell cancer therapy. Its inhibition would suppress cystine uptake by certain progressed cells, and also interfere with cystine uptake, and subsequent cysteine release, by eg macrophages, thought to have a role in cysteine delivery to lymphoid cells.

Animals↗
PubMed1996

A comparative study of the effects of timolol and latanoprost on blood flow velocity of the retrobulbar vessels.

PURPOSE: To examine the effects of topical timolol and latanoprost on retrobulbar vessel blood velocity in patients with glaucoma or ocular hypertension. METHODS: Nine patients with primary open-angle glaucoma and six patients with ocular hypertension were enrolled for this study. All patients were treated topically with 0.5% timolol or 0.005% latanoprost, using a double-masked crossover design with a 3-week washout before administration of each drug. Each patient had a baseline color Doppler imaging ultrasound of the central retinal artery, short posterior ciliary arteries, and ophthalmic artery and two other ultrasound examinations during the 1-week treatment with each drug, performed 12 hours after the first dose of the drug and 12 hours after the last dose, 7 days later. RESULTS: Both topical timolol and topical latanoprost significantly reduced the intraocular pressure. The only significant change observed in the retrobulbar blood velocity with timolol was a reduction of end diastolic velocity in the ophthalmic artery 12 hours after the first dose, accompanied by a trend toward a decrease in the peak systolic velocity and an increase in the resistance index in the same vessel. No change in blood velocity was observed with latanoprost. CONCLUSION: Topical timolol and latanoprost significantly reduced the intraocular pressure in ocular hypertensive and glaucoma patients without creating substantial hemodynamic changes in the retrobulbar vessels.

Administration, Topical↗
PubMed1996

Color Doppler imaging in patients with asymmetric glaucoma and unilateral visual field loss.

PURPOSE: To determine whether lower blood velocities and high resistive index in the retrobulbar arteries are primary or secondary to glaucomatous damage in patients with open-angle glaucoma. METHODS: Color Doppler imaging was performed in 32 glaucomatous patients with unilateral visual field loss and in 31 control subjects. Peak systolic velocity and end diastolic velocity were measured, and resistive index was calculated in the central retinal artery and short posterior ciliary arteries. RESULTS: In patients with glaucoma, both the more affected and the contralateral eyes with normal visual fields had significantly lower peak systolic velocity and end diastolic velocity in their central retinal artery and short posterior ciliary arteries than did the control subjects of similar age (P < or = .03). The resistive index of the central retinal artery of both eyes of patients with glaucoma was also significantly higher than in the control subjects (P = .001). When considering the 16 patients who had the greatest visual field asymmetry, the more affected eyes had lower peak systolic velocity and end diastolic velocity in the central retinal artery than the contralateral eyes did (P = .02). CONCLUSIONS: Even eyes with normal visual fields in patients with asymmetric disease had decreased blood velocities in their retrobulbar vessels, suggesting that these circulatory changes probably precede detectable damage. Furthermore, the finding of lower central retinal artery blood velocities in the more affected eye of asymmetric patients suggests that low blood velocities may be one of the lateralizing factors in those patients and that they have a possible role in the pathogenesis of the disease.

Aged↗
PubMed1996

Various glaucomatous optic nerve appearances. A color Doppler imaging study of retrobulbar circulation.

PURPOSE: The purpose of this study was to observe the blood flow velocity in the retrobulbar vessels of patients with glaucoma with different appearances of optic discs. METHODS: Patients with four different disc appearances (focal ischemic discs, myopic glaucomatous discs, senile sclerotic discs, and discs with a generalized enlargement of the cup) were selected from a pool of optic disc photographs. No clinical information from the patients was available during the selection. Color Doppler imaging (CDI) was performed in those patients whose discs clearly belonged to one of the disc patterns. The peak systolic and end diastolic velocity were measured and the resistance index calculated from the central retinal artery, short posterior ciliary arteries and ophthalmic artery. RESULTS: Color Doppler imaging was performed in 24 patients with focal ischemic discs, 26 patients with myopic glaucomatous discs, 16 patients with senile sclerotic discs, and 16 patients with generalized enlargements of the cup. The patients with senile sclerotic discs had statistically significantly lower diastolic velocity and a higher resistance index in their ophthalmic, central retinal and the mean of the short posterior ciliary arteries sampled. These statistical differences persisted for the ophthalmic artery even after correcting the values for the age differences between the groups. CONCLUSIONS: Patients with senile sclerotic discs seem to have greater circulatory abnormalities in their retrobulbar vessels studied with color Doppler imaging that are compatible with a higher downstream resistance in these vessels. These circulatory abnormalities could be related to the pathogenesis of the glaucoma in these patients.

Aged↗
PubMed1996

Rapid modulation of the apoptosis regulatory genes, bcl-2 and bax by prolactin in rat Nb2 lymphoma cells.

The Nb2 rat lymphoma represents a useful model for investigation of molecular events coupled to PRL-induced proliferation. Moreover, recent evidence has also demonstrated the utility of this system to study mechanisms linked to programmed cell death (apoptosis). Thus, glucocorticosteroids activate apoptosis in lactogen-dependent Nb2 cells, whereas the addition of PRL abrogates this effect. The present study was conducted to determine whether PRL stimulation in lactogen-dependent Nb2-11 or autonomous Nb2-SFJCD1 cultures alters expression of bcl-2 or bax, genes that suppress or facilitate apoptosis, respectively. We demonstrate that PRL stimulation in stationary Nb2-11 cultures significantly increased the level of bcl-2 messenger RNA (mRNA) within 3 h (15-fold) and its protein product by 6 h, time points previously shown to correspond with G1 cell cycle progression. In Nb2-SFJCD1 cells, bcl-2 mRNA was found to be constitutively present. Addition of PRL to these lactogen-independent cultures further enhanced its expression at the mRNA and protein levels with a kinetic pattern similar to that observed in the PRL-dependent line. Results from stability studies indicated that increased bcl-2 mRNA evoked by PRL in Nb2 cell lines was most likely not attributable to increased stability of its transcripts. Furthermore, the rapid increase in its expression in PRL-stimulated Nb2-11 cells was not altered by inhibition of protein synthesis suggesting a direct action of the hormone. PRL also increased bax mRNA by 8 h in Nb2-11 cultures. However, hormone stimulation markedly attenuated the level of the Bax protein from 2-6 h. In contrast, bax expression in Nb2-SFJCD1 cultures remained unaltered by the addition of the hormone. These results demonstrate that altered expression of bcl-2 and bax are each associated with PRL-stimulated cell cycle progression in Nb2 cells. Moreover, bcl-2 appears to be an immediate-early gene induced by PRL in the hormone-dependent line and may represent an important regulator of early G1 cell cycle progression most likely by suppressing cell death mechanisms.

Animals↗
PubMed1996

Visual field correlations with color Doppler studies in open angle glaucoma.

PURPOSE: This study was designed to assess the correlation between flow velocity, the resistive index and visual field defects in patients with chronic open angle glaucoma in comparison with a nonglaucomatous control population. METHODS: Color Doppler imaging was used to study flow velocity in the central retinal artery and short posterior ciliary arteries of 76 patients with chronic open angle glaucoma and 28 normal subjects. Velocity and resistive index were correlated with visual field abnormalities. RESULTS: The chronic open angle glaucoma patients showed a statistically significant lowering of the end diastolic velocity and a raised resistive index in all vessels studied. The end diastolic velocity of the central retinal arteries of glaucoma patients were significantly correlated with the Mean Deviation of the visual field (right eye, p = 0.0041; left eye, p = 0.0167). The glaucoma patients showed a statistically significant lowering of the end diastolic velocity and a raised resistive index in all vessels supplying those parts of the optic disc that corresponded to visual hemifield defects. CONCLUSION: Open angle glaucoma is associated with changes in central retinal and ciliary artery flow velocity and resistive index which suggest a compromised circulation in this region.

Adult↗
PubMed1996

Ocular hypertension and primary open-angle glaucoma: a comparative study of their retrobulbar blood flow velocity.

PURPOSE: To compare the orbital blood flow velocities of patients with long-standing ocular hypertension and patients with primary open-angle glaucoma. METHODS: Twenty patients with ocular hypertension were recruited from our clinic and underwent color Doppler imaging evaluation of their retrobulbar vessels. The blood flow velocities and resistance index of their central retinal artery, temporal short posterior ciliary artery, and ophthalmic artery were compared with those of 20 glaucoma patients individually matched for age and level of the highest untreated intraocular pressure ever recorded. RESULTS: Glaucoma patients had significantly lower peak systolic velocity and end-diastolic velocity than did patients with ocular hypertension in their central retinal artery (p < 0.001). No significant difference between the groups was observed in the other vessels studied. CONCLUSIONS: Glaucoma patients had lower blood flow velocity in the central retinal artery compared with that of ocular hypertension patients of similar age and level of untreated intraocular pressure. This might be important in the development of glaucomatous damage in those patients.

Blood Flow Velocity↗
PubMed1996

Alterations in pim-1 and c-myc expression associated with sodium butyrate-induced growth factor dependency in autonomous rat Nb2 lymphoma cells.

Whereas Nb2-11 lymphoma cells critically require prolactin (PRL) for growth, Nb2-SFJCD1 subline cells are growth factor independent. Treatment with the differentiating agent, sodium butyrate (NaBT), has been demonstrated previously to lead to growth arrest of Nb2-SFJCD1 cells and a transient reversion to PRL growth requirement following removal of NaBT. In the present study, the relation of NaBT-induced growth arrest to the cell cycle was examined using flow cytometry, and the effect of PRL on expression of the immediate-early proto-oncogenes, pim-1 and c-myc, in NaBT-pretreated cultures was evaluated. Treatment of Nb2-SFJCD1 cells with 2 mM NaBT for 72 h caused growth arrest in the majority of the cells in the G1 phase of the cell cycle, an effect similar to that produced by lactogen deprivation in PRL-dependent Nb2 cultures. The addition of PRL stimulated a concentration-dependent re-entry into the cell cycle. In other experiments, NaBT treatment significantly reduced the steady-state levels of pim-1 and c-myc mRNA. Stimulation with PRL induced a rapid and concentration-dependent biphasic accumulation of each mRNA with similar kinetics. Maximal expression of both proto-oncogenes occurred within 2-4 h and after 12 h. Results from mRNA stability studies suggest that the observed increases in expression of pim-1 and c-myc most likely do not reflect increased stability of the transcripts. The results indicate that NaBT-induced differentiation in autonomous Nb2-SFJCD1 causes growth arrest of the cells in the G1 phase of the cell cycle and reduces the basal levels of pim-1 and c-myc mRNAs. Mitogenic stimulation with PRL reinitiates cell cycle progression characterized by biphasic expression of each proto-oncogene. It is suggested that NaBT is a useful tool for investigation of the malignant progression from growth factor dependency to autonomy in the Nb2 lymphoma paradigm.

Animals↗
PubMed1995

A novel heat shock response in prolactin-dependent Nb2 node lymphoma cells.

Virtually all cells respond to heat stress by increased expression or induction of one or more of the highly conserved cellular stress response proteins, heat shock proteins (HSPs). Here, we report the unusual property of rat Nb2-11 cells, a prolactin-dependent pre-T-cell line, to display reduced HSP expression following exposure to elevated temperature. After heat stress (41 degrees C, 1 h), there was no evidence of inducible members of the 70 kDa HSP family, a response common to other cell culture and tissue systems. Moreover, expression of constitutive members of the HSP70 and HSP90 families decreased during the heat stress, apparently reflecting a decrease in mRNA stability. Gel shift assays revealed that heat shock factor (HSF) was activated in spite of the lack of expression of inducible HSP70 transcripts, although its DNA binding rapidly deteriorated. Immunoblotting, using an antibody specific to HSF1, indicated that proteolysis of HSF1 may be responsible for this rapid termination of heat shock element binding. CCAAT binding, a component of constitutive HSP70 expression, was also reduced by heat stress in Nb2-11 cells and may account for the decline in constitutive HSP70 expression. Prolactin pretreatment prevented the fragmentation of HSF1, protected heat shock element and CCAAT binding, prevented the decline in constitutive HSP70 and HSP90 expression, and restored a modest expression of inducible HSP70 following heat treatment. Results of this study describe a unique regulatory defect in HSP expression in Nb2-11 cells, possibly a common characteristic of other hormone-dependent tumors.

Animals↗
PubMed1995

The nuclear prolactin receptor: a 62-kDa chromatin-associated protein in rat Nb2 lymphoma cells.

Previously we demonstrated that lactogen-dependent Nb2 cells express a nuclear prolactin (PRL) receptor. Thus, the nuclear receptor expressed in PRL-dependent Nb2-11 and -independent Nb2-SFJCD1 cells was characterized. Initially, the potential proteolytic processing of internalized 125I-rPRL was investigated. Radiolabeled hormone eluted from a Sephadex G-100 column with a retention time identical to that found for stock hormone, indicating that nuclear PRL was intact. Experiments to investigate the nuclear distribution of the hormone demonstrated that 90% of 125I-rPRL bound to chromatin; the remaining was distributed between "sap-protein" and nucleoplasmic fractions. Chromatin-bound PRL was resistant to high salt and detergent extraction indicating a tight association. Immunoprecipitation and immunoblot analysis revealed the PRL receptor to be 62 kDa in each cell line. Affinity crosslinking experiments and immunoprecipitation demonstrated that 125I-rPRL complexed with a protein(s) of similar M(r) in intact cells. 125I-rPRL binding was saturable and of high affinity (Kds of 180 and 170 pM, for Nb2-11 and Nb2-SFJCD1 lines, respectively). PRL binding was competitively inhibited by ovine and bovine PRLs and hGH, but not by rat GH, and by monoclonal antibodies (McAbs) which recognize the lactogen binding site. These results demonstrate that: (1) 125I-rPRL translocates intact to the Nb2 cell nucleus and tightly associates with chromatin; (2) the chromatin receptor specifically binds 125I-rPRL with high affinity; and (3) the chromatin receptor is essentially identical to its membrane counterpart with respect to mass, binding characteristics, and McAb recognition.

Animals↗