Am J Physiol Cell Physiol AJP: Cell Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 295: C406-C413, 2008. First published June 11, 2008; doi:10.1152/ajpcell.00450.2007
0363-6143/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/2/C406    most recent
00450.2007v1
Right arrow Alert me when this article is cited
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
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sanders, J. A.
Right arrow Articles by Gruppuso, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sanders, J. A.
Right arrow Articles by Gruppuso, P. A.

GROWTH, DIFFERENTIATION, AND APOPTOSIS

The effect of rapamycin on DNA synthesis in multiple tissues from late gestation fetal and postnatal rats

Jennifer A. Sanders, Alisha Lakhani, Chanika Phornphutkul, Ke-Ying Wu, and Philip A. Gruppuso

Division of Pediatric Endocrinology and Metabolism, Rhode Island Hospital, and the Warren Alpert Medical School of Brown University, Providence, Rhode Island

Submitted 28 September 2007 ; accepted in final form 9 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Rapamycin is a potent antiproliferative agent that arrests cells in the G1 phase of the cell cycle through a variety of mechanisms involving the inhibition of the mammalian target of rapamycin (mTOR) pathway. The majority of normal cells in culture are sensitive to the cytostatic effects of rapamycin, whereas the growth of many malignant cells and tumors is rapamycin resistant. We had shown previously that hepatic DNA synthesis in the late gestation rat fetus is rapamycin resistant even though signaling through the mTOR/S6 kinase (S6K) pathway is attenuated. On the basis of this finding, we went on to characterize the response to rapamycin in a spectrum of tissues during late gestation and the early postnatal period in the rat. We found that rapamycin had no effect on DNA synthesis in major organs such as heart, intestine, and kidney in the fetal and early postnatal rat despite a marked attenuation in the phosphorylation of ribosomal protein S6. In contrast, the proliferation of mature hepatocytes during liver regeneration was highly sensitive to rapamycin. These data indicate that basal cellular proliferation in a wide variety of tissues is rapamycin resistant and occurs independently of mTOR/S6K signaling. Furthermore, the well-characterized effects of rapamycin in tissue culture systems are not recapitulated in the asynchronous cell proliferation that accompanies normal growth and tissue remodeling.

proliferation; mammalian target of rapamycin; growth

THE MACROLIDE ANTIBIOTIC, rapamycin, was first identified as an anti-fungal agent produced by Streptomyces hygroscopicus. It was later discovered to have numerous potent immunosuppressive and antitumor activities (27, 28). The target of rapamycin (TOR) was originally identified in the yeast Saccharomyces cerevisiae using a screen for mutants that were able to proliferate in the presence of rapamycin. Rapamycin does not directly inhibit TOR. Instead, it forms a complex with FK506 binding protein (FKBP12), and this complex then binds to TOR, inhibiting its serine threonine kinase activity. The mammalian homologue of yeast TOR, mTOR, was identified on the basis of its ability to bind the rapamycin-FKBP12 complex. Similar to its role in yeast, mTOR is responsive to nutrients and plays a fundamental role in coupling nutrient availability to the regulation of cell growth (size) and proliferation (2, 17).

To couple cellular growth and proliferation to energy status and nutrient availability, control of the production and abundance of ribosomes must be tightly regulated. Two of the critical downstream targets of mTOR involved in protein translation and ribosome biogenesis are S6 kinase (S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1; 10, 14). mTOR phosphorylation of S6 kinase contributes to increased kinase activity and elevated phosphorylation of ribosomal protein S6. This phosphorylation of S6 has been reported to selectively increase the translation of a subset of mRNAs with a 5' pyrimidine tract (TOP) motif. Other studies have shown that deletion of both S6K1 and S6K2 or mutation of the phosphorylation sites of ribosomal protein S6 does not affect the translational control of 5' TOP mRNAs, calling into question the physiological role of S6 (25, 26). Many of these TOP-containing mRNAs encode ribosomal proteins, elongation factors, and other components of the translation apparatus (9). 4EBP1, another well-characterized target of mTOR, acts as a translation repressor when hypophosphorylated. It does so by binding and inhibiting the eukaryotic translation initiation factor 4E (eIF4E), which recognizes the 5' end cap of eukaryotic mRNAs. Phosphorylation of 4EBP1 by mTOR results in the release of eIF4E and an increase in cap-dependent translation. Rapamycin has been shown to inhibit the phosphorylation of S6 kinase and 4EBP1 by mTOR, thus inhibiting mTOR-mediated activation of the cell's translational capacity (8, 18).

In addition to its role in protein translation and ribosome biogenesis, mTOR has been shown to regulate the levels of several proteins directly involved in the cell cycle (21). In many cell systems, rapamycin treatment has been shown to inhibit the expression of D-type cyclins and to suppress c-Myc translation (13). Cyclin A and PCNA expression are also suppressed by rapamycin, although the mechanisms involved are unclear (21, 30). In addition, rapamycin has been shown to inhibit the induction of cyclin E expression in NIH3T3 cells, implicating the mTOR/S6 kinase pathway in the regulation of cyclin E (6).

The cyclin-dependent kinase inhibitors p21 and p27 are also downstream effectors of rapamycin action. The role of p21 and p27 in rapamycin action is complex and cell type-specific, depending on the degree to which p21 and p27 act as inhibitors or activators of the relevant cyclin-dependent kinases (21). In T cells, rapamycin inhibition of mitogenesis is associated with decreased expression of p21 (19). A similar effect of rapamycin has been reported in mouse fibroblasts (11). In these cells, treatment with rapamycin resulted in a reduction in the activity of both cyclin D1/cdk4 and cyclin E/cdk2 complexes. The loss in activity was associated with a reduction in the total level of p21 and in the amount of p21 associated with cyclin D1/cdk4 complexes. Rapamycin has also been shown to induce cell cycle arrest by increasing p27 levels (5, 22). The mechanisms by which rapamycin increases p27 levels varies according to cell type and includes regulation at the transcriptional, translational, and posttranslational levels (2, 21, 29, 34).

Our prior studies on mitogenic signaling and rapamycin sensitivity during liver development in the rat have shown that the pathways that mediate mitogen-stimulated hepatocyte proliferation in the adult rat are not active in the late gestation fetus. Our data have shown that in late gestation, the ERK1/2 MAPK pathway is uncoupled, insulin signaling through the Akt/mTOR/S6K pathway is attenuated, and hepatocyte proliferation is resistant to rapamycin (1, 3, 4). In the current study, we investigated the effect of rapamycin on the mTOR/S6K pathway and cellular proliferation in a spectrum of tissues in fetal and early postnatal rats to test the hypothesis that the rapamycin resistance of late gestation fetal hepatocytes was a unique characteristic of these cells. Our results refute this hypothesis, raising questions about the applicability of available data on mTOR signaling to the cell proliferation that occurs as a component of normal growth and tissue remodeling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male and timed-pregnant female Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA). Rats were fed standard laboratory chow ad libitum and were killed under pentobarbital anesthesia. For fetal studies, a laparotomy was performed under pentobarbital anesthesia (40 mg/kg ip injection) on embryonic day 19 (E19). Fetuses were exteriorized under sterile conditions. Rapamycin (5.0 µg/g or 10.0 µg/g; LC Laboratories, Woburn, MA) or an equivalent amount of DMSO vehicle was administered by intraperitoneal injection in situ. 5'-Bromo-2'-deoxyuridine (BrdU) was coadministered to all fetuses (60 µg/g estimated body wt; Sigma, St. Louis, MO). Body weight was estimated at 2 g for E19 fetuses. The fetuses were replaced, laparotomy incisions were closed, and gestation was allowed to continue for 24 h, at which time Cesarean sections were performed to obtain the fetuses. Fetuses were rapidly weighed before being fixed in 10% neutral-buffered formalin.

For postnatal studies, 10-day-old (P10) rats were derived from dams of known gestational age, which were allowed to deliver spontaneously and remain with their offspring. P10 rats were injected daily with DMSO vehicle or rapamycin by intraperitoneal injection as for the fetal studies. Again, BrdU was coadministered. Animals were killed on postnatal day 13.

Two-thirds partial hepatectomy was performed under isoflurane anesthesia on adult male rats (125–150 g). DMSO vehicle or 2.5 µg/g rapamycin were coadministered with 60 µg/g BrdU by intraperitoneal injection 1 h before surgery. Rats received daily intraperitoneal injections of BrdU plus DMSO vehicle or rapamycin and were killed 72 h posthepatectomy. All animal studies were approved by the Rhode Island Hospital Animal Care and Use Committee and were in accordance with the criteria outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, Revised 1996).

Sample preparation. Tissue homogenates were prepared using 20 ml/g tissue of buffer A (10 mM Tris, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM Na pyrophosphate, 50 mM Na fluoride, and 100 µM Na orthovanadate) containing protease inhibitors [10 mg/ml leupeptin, 10 mg/ml aprotinin, and 34.4 mg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride]. Following homogenization, Triton X-100 was added to a final concentration of 1%, followed by incubation on ice for 30 min. The homogenates were centrifuged at 1,000 g for 15 min. The resulting supernatant was centrifuged at 6,800 g for 20 min. An aliquot of the final supernatant was used to determine the protein concentration using the bicinchoninic acid method. The remaining supernatant was boiled in Laemmli sample buffer for 10 min and frozen at –70°C until use.

Western immunoblot analysis. Samples were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and subjected to immunoblot analysis with antibodies directed toward ribosomal protein S6 or phosphorylated S6Ser235/236 (P-S6Ser235/236). Both antibodies were obtained from Cell Signaling Technology (Beverly, MA). Immunoblotting for the p85 subunit of phosphoinositide 3-kinase (PI3K) was used as a loading/transfer control. The p85 antibody was obtained from Upstate (Lake Placid, NY). Detection used enhanced chemiluminescence. Equal amounts of protein were loaded in each lane on the basis of protein determinations performed using the bicinchoninic acid method.

Immunohistochemistry. E20 fetuses, metatarsals, and tissues from P10 and adult rats were fixed in 10% neutral-buffered formalin, embedded in paraffin, and analyzed by hematoxylin and eosin staining and immunohistochemistry for BrdU, Ki-67, and P-S6235/236 using the indirect immunoperoxidase technique. Briefly, 6-µm paraffin sections were passed through xylene, graded alcohol, and rinsed in phosphate-buffered saline. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide. Slides were incubated in 0.1% Protease XIV in 0.5 M Tris, pH 7.5, for 5 min at 37°C, followed by denaturation in 2 N HCl for 30 min at 37°C and neutralization in 0.1 M Borax, pH 8.5, for 5 min before overnight incubation at 4°C in the anti-BrdU antibody (Vector Labs, Burlingame, CA). Fetuses that had not been injected with BrdU were used as a negative control. For Ki-67 staining, slides were microwaved in 100 mM sodium citrate buffer, pH 6.0, for 15 min, followed by a 30-min incubation at 37°C in anti-Ki-67 antibody (Dako, Carpinteria, CA). BrdU or Ki-67 positive cells in three x20 fields in three sections from duplicate animals were counted. Detection of P-S6235/236 used a rabbit antibody (Cell Signaling Technology) without antigen retrieval in an overnight incubation at 4°C. For all antibodies, detection steps used a horseradish-peroxidase conjugated secondary antibody. Signal was detected using the ABC elite kit and 3,3-diaminobenzidine (Vector Labs). Metatarsals and fetuses were counterstained with hematoxylin AS (Vector Labs). Omission of primary antibody was used as a control to test for the specificity of staining. Slides were viewed and digital images acquired with a Nikon E800 microscope and Spot II digital camera.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of rapamycin on hepatic DNA synthesis. Our initial experiments were aimed at extending our previous findings on the rapamycin sensitivity of late gestation fetal and regenerating liver (3). In our previous studies, rapamycin significantly reduced BrdU incorporation in 24-h regenerating liver (3). The fetal studies were repeated, again demonstrating that rapamycin administration to E19 fetuses in situ had no effect on BrdU incorporation over the ensuing 24 h (Fig. 1). Hepatocyte proliferation after two-thirds partial hepatectomy is synchronous with the majority of the cells reaching S phase at 24 to 36 h (31). In the current study, we chose to study 72-h regenerating liver to obtain the maximal effect of rapamycin on BrdU incorporation during liver regeneration. In contrast with late gestation fetal liver, administration of rapamycin before two-thirds partial hepatectomy induced significant inhibition of adult hepatocyte proliferation that was sustained for up to 72 h (Fig. 1, left). To confirm the results obtained using BrdU labeling, we used Ki-67 as another marker for cellular proliferation (Fig. 1, right). In accordance with our BrdU incorporation data, Ki-67 staining was unchanged in rapamycin-treated fetuses compared with vehicle, whereas a marked decrease in staining for Ki-67 was observed in 72-h regenerating liver. These results indicate that, in contrast with late gestation hepatocytes, the mTOR/S6 kinase signaling pathway is required for hepatocyte proliferation during early liver regeneration.


Figure 1
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 1. Effect of rapamycin on DNA synthesis in fetal and adult liver. Fetuses on embryonic day 19 (E19) were injected in situ with DMSO vehicle or rapamycin plus 5'-bromo-2'-deoxyuridine (BrdU). The fetuses were replaced, and gestation was allowed to continue for 24 h. Adult rats were injected with DMSO vehicle or rapamycin plus BrdU prior to 2/3 hepatectomy (PH). Livers were harvested 72 h after surgery. Formalin-fixed, paraffin-embedded liver sections were stained for BrdU (left) or Ki-67 (right). Graphs show total number of positive nuclei per three x20 fields for duplicate animals ({circ}, vehicle; bullet, rapamycin). Data are shown as individual circles with the mean.

 
Fetal DNA synthesis is rapamycin resistant. Our previous findings of rapamycin-insensitive cell proliferation in fetal liver led us to question whether other fetal tissues are rapamycin resistant. To address this issue, we administered rapamycin or vehicle in combination with BrdU by intraperitoneal injection to E19 fetuses in situ and performed BrdU immunohistochemistry on whole mount sections (Fig. 2A). The results we obtained were unexpected. A spectrum of tissues, including stomach, intestine, lung, kidney, and heart, showed no effect of rapamycin on BrdU incorporation 24 h after the initial injection. Fetal weight used as an indicator of fetal size was also unaffected by rapamycin (Fig. 2B).


Figure 2
View larger version (45K):
[in this window]
[in a new window]

  		Fig. 2. Effect of rapamycin on DNA synthesis in E19 fetal rats. E19 fetuses were injected in situ with DMSO vehicle or rapamycin plus BrdU. The fetuses were replaced, and gestation was allowed to continue for 24 h. Control fetuses were not injected with BrdU and were harvested on embryonic day 20. A: fetal whole mounts were fixed in formalin, paraffin-embedded, and stained for BrdU. B: illustration of fetal body weight from litters injected in situ with DMSO vehicle (open bar) or rapamycin (filled bar). Data are means + 1 SD. The data represent analysis of the means from three litters.

 
The absence of an obvious effect of rapamycin on BrdU incorporation in major organ systems could be due to limited bioavailability of the rapamycin. To confirm the bioavailability and bioactivity of rapamycin, Western immunoblot analysis was performed for phosphorylated and total ribosomal protein S6. Staining for P-S6235/236 was abolished by rapamycin treatment in both fetal and regenerating liver (Fig. 3A). We also examined the ability of a single intraperitoneal dose of rapamycin to inhibit mTOR/S6K1 signaling for the 24-h duration of the experiment in major organ systems. Immunohistochemistry for P-S6235/236 (Fig. 3B) showed strong cytoplasmic staining of skin and metatarsal bones in vehicle-injected fetuses. Administration of rapamycin resulted in a profound inhibition of S6 phosphorylation at these two sites. The majority of the other tissues showed weak cytoplasmic staining for P-S6235/236 in vehicle-injected fetuses that was abolished by rapamycin (data not shown). Given the relatively weak staining in vehicle-treated fetuses, we confirmed the efficacy of rapamycin in several of the tissues by performing Western immunoblot analysis for total S6 and P-S6235/236 (Fig. 3C). In agreement with our immunohistochemical staining, rapamycin induced a significant inhibition of P-S6235/236 in kidney. Similar results were obtained in homogenates prepared from intestine (data not shown).


Figure 3
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 3. Effect of rapamycin on signaling through the mammalian target of rapamycin/S6 kinase (mTOR/S6K) pathway. Liver homogenates were prepared from E19 and regenerating liver (PH), separated by SDS-polyacrylamide gel electrophoresis, and analyzed for phosphorylated S6Ser235/236 (P-S6235/236) and total S6. A: Western immunoblotting for the p85 catalytic subunit of phosphoinositide 3-kinase (PI3K) was used as a loading/transfer control. V, vehicle; R, rapamycin. B: immunohistochemistry on formalin-fixed, paraffin-embedded fetuses and dissected metatarsals stained for P-S6235/236 and counterstained with hematoxylin. C: total kidney homogenates were analyzed by Western immunoblotting for P-S6235/236, total S6, and the p85 catalytic subunit of PI3K.

 
Having determined that a single injection of rapamycin was sufficient to cause a profound and persistent inhibition of S6 phosphorylation, we went on to quantify the effect of rapamycin on DNA synthesis in kidney, intestine, and metatarsals from vehicle- and rapamycin-injected fetuses by quantifying BrdU-positive nuclei in duplicate samples (Fig. 4). Despite the potent effect of rapamycin on S6 phosphorylation, there was no discernible effect on tissue morphology, cell size, or DNA synthesis after 24 h in any of the tissues examined. To confirm this result, we performed Ki-67 staining to identify cells in the S-phase of the cell cycle. In accordance with the BrdU results, kidney (Fig. 5), intestine, and liver (data not shown) displayed high levels of proliferation in animals injected with either vehicle or rapamycin. The number of Ki-67- positive cells in rapamycin-injected fetuses was not decreased compared with those that were administered vehicle. The studies were repeated using a higher concentration of rapamycin (10 µg/fetus). Increasing the dose of rapamycin had no effect on fetal weight, BrdU incorporation, or Ki-67 staining (data not shown). We interpreted these results as indicating that rapamycin had no cell cycle inhibitory effect in late gestation kidney, intestine, and metatarsal as any period of inhibition would have caused a decrease in the BrdU-labeling index or Ki-67 staining.


Figure 4
View larger version (81K):
[in this window]
[in a new window]

  		Fig. 4. Effect of rapamycin on DNA synthesis in multiple E19 fetal tissues. E19 fetuses were injected in situ by intraperitoneal injection with vehicle or rapamycin plus BrdU. The fetuses were replaced, and gestation was allowed to continue for 24 h. Fetuses and dissected metatarsals were fixed in formalin, paraffin-embedded, and stained with hematoxylin and eosin (A) or BrdU (B). Metatarsals were counterstained with hematoxylin in B. The graph shows total number of positive nuclei per three x20 fields for duplicate animals ({circ}, vehicle; bullet, rapamycin). Data are shown as individual circles with the mean.

 

Figure 5
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 5. Effect of rapamycin on cellular proliferation in E19 fetal kidney. E19 fetuses were injected in situ by intraperitoneal injection with vehicle or rapamycin plus BrdU. The fetuses were replaced, and gestation was allowed to continue for 24 h. Fetuses were fixed in formalin, paraffin-embedded, and stained for Ki-67. Representative sections of kidney are shown. The graph shows total number of positive nuclei per three x20 fields for duplicate animals ({circ}, vehicle; bullet, rapamycin). Data are shown as individual circles with the mean.

 
Postnatal DNA synthesis is insensitive to rapamycin. Our prior studies have demonstrated that the signaling pathways commonly associated with cellular proliferation and growth, the ERK1/2 and PI3K-mTOR pathways, are attenuated in late gestation fetal liver but become growth factor responsive during the first postnatal week (1, 4). Given the change in PI3K-mTOR signaling, we hypothesized that basal cellular proliferation in early postnatal animals would be rapamycin sensitive. To test this hypothesis, we conducted a series of experiments in 10-day-old animals to assess the postnatal effect of rapamycin on DNA synthesis in liver, kidney, and intestine.

As was the case in the late gestation fetus, we observed no effect of rapamycin on tissue morphology, cell size, or DNA synthesis in these tissues as assessed by hematoxylin and eosin staining and BrdU incorporation (Fig. 6). As in the fetal tissues, Ki-67 was used as an additional marker of cellular proliferation. In accordance with the BrdU data, Ki-67 staining was unaffected by rapamycin in both liver and kidney (Fig. 7). To confirm the potency of the injected rapamycin, Western immunoblotting for total S6 and P-S6235/236 was performed in total homogenates from these tissues (Fig. 8). Again, these results were interpreted as indicating that the DNA synthesis associated with basal levels of postnatal cell proliferation is rapamycin resistant despite the potent inhibitory effect of the drug on mTOR/S6K signaling.


Figure 6
View larger version (65K):
[in this window]
[in a new window]

  		Fig. 6. Effect of rapamycin on DNA synthesis in early postnatal rats. Rat pups were injected with DMSO vehicle or rapamycin plus BrdU by intraperitoneal injection on days 10, 11, and 12 of life. Liver, kidney, and intestine were harvested on postnatal day 13. Representative formalin-fixed, paraffin-embedded sections of liver, kidney, and intestine were stained with hematoxylin and eosin (A) or BrdU (B). The graph shows total number of positive nuclei per three x20 fields for duplicate animals ({circ}, vehicle; bullet, rapamycin). Data are shown as individual circles with the mean.

 

Figure 7
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 7. Effect of rapamycin on cellular proliferation in early postnatal rat liver and kidney. Rat pups were administered DMSO vehicle or rapamycin plus BrdU by intraperitoneal injection on days 10, 11, and 12 of life. Liver, kidney, and intestine were harvested on postnatal day 13. Formalin-fixed, paraffin-embedded sections of liver and kidney were stained for Ki-67. The graph shows total number of positive nuclei per three x20 fields for duplicate animals ({circ}, vehicle; bullet, rapamycin). Data are shown as individual circles with the mean.

 

Figure 8
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 8. Effect of rapamycin on mTOR/S6K signaling in early postnatal rats. Total kidney homogenates were prepared, separated by SDS-polyacrylamide gel electrophoresis, and analyzed for P-S6235/236 and total S6. Western immunoblotting for the p85 catalytic subunit of PI3K was used to control for loading and the transfer.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In many different cell types, rapamycin has been shown to induce G1 growth arrest in vitro by a variety of mechanisms (16, 20, 28). Few data are available on the in vivo effects of rapamycin on cell proliferation. In the present study, we found that cellular proliferation in a spectrum of tissues in the late gestation and early postnatal rat were resistant to the antiproliferative effect of rapamycin. Fetal weight, which is an indicator of cell size, was also unaffected by rapamycin. At both of these developmental ages, rapamycin had a potent inhibitory effect on the mTOR/S6 kinase pathway, as manifested as a loss of S6 phosphorylation. Previous studies in our laboratory have shown that both S6K1 and S6K2 activities are inhibited in response to rapamycin in both fetal and regenerating liver (3). However, adult hepatocyte proliferation during liver regeneration was highly sensitive to the antiproliferative effect of rapamycin. These studies suggest that under basal conditions in the late gestation and early postnatal rat, cell proliferation in multiple tissues can occur independent of mTOR/S6K pathway activation. In contrast, this pathway is necessary for the synchronized hepatocyte proliferation that occurs following two-thirds partial hepatectomy. These results also indicate that the intrinsic resistance to rapamycin observed in the fetal and early postnatal tissues cannot be accounted for by differences in the effect of rapamycin on the mTOR/S6 kinase signaling cascade.

Potent inhibition of the mTOR/S6 kinase pathway has been reported in multiple rapamycin-resistant cell lines (12, 15, 22). In many of these lines, rapamycin resistance has been correlated with differential regulation of p27 and/or c-myc. In highly resistant rhabdomyosarcoma cell lines, intrinsic and acquired resistance to rapamycin was found to correlate with the induction or continued expression of c-myc (12, 15). However, in both the fetal and regenerating liver, rapamycin had no effect on c-Myc protein levels (J. Sanders and P. Gruppuso, unpublished observations), suggesting that, in hepatic cells in vivo, rapamycin resistance is not mediated by changes in c-myc regulation. In other cases, the response to rapamycin has been tied to the regulation of p27 (16, 23, 33). In sensitive cells, the antiproliferative effects of rapamycin have been correlated with an inhibition of the mitogen-stimulated downregulation of p27. Furthermore, p27-null fibroblasts and T cells derived from p27–/– mice were shown to be resistant to the growth inhibitory effects of rapamycin (22). However, in another study conducted by Nakayama et al. (24), p27-null T cells were shown to be sensitive to rapamycin. We have found evidence that rapamycin-induced cell cycle arrest can occur through multiple mechanisms across various hepatic cell lines and between fetal and adult liver. In some cases, p27-mediated inhibition of cyclin E-dependent kinase activity seems important, whereas in other cell lines, neither p21 nor p27 seems to be involved (J. Boylan and P. Gruppuso, unpublished observations).

Deregulation of eIF4E has also been widely recognized as a mechanism for rapamycin resistance in cancer cells (8, 20, 22). Dilling et al. (8) found that rapamycin-insensitive colon carcinoma cell lines exhibited low 4EBP:eIF4E ratios and that overexpression of 4EBP in these cells resulted in a marked increase in rapamycin sensitivity. Other studies have reported that eIF4E overexpression promotes tumor progression and rapamycin resistance (7, 20, 32). However, in contrast to the case in cultured cells, regulation of the formation of the cap-binding complex was shown to be rapamycin insensitive in regenerating liver, suggesting that control of eIF4E via effects on 4EBP may not account for the differential effects of rapamycin on DNA synthesis in fetal versus adult liver (19).

The mechanisms for rapamycin resistance are complicated, numerous, and appear to vary depending on the cell system employed. Regardless of the exact mechanism for rapamycin resistance, our findings lead to several conclusions. The first is that, under basal conditions, DNA synthesis in many fetal and early postnatal tissues is independent of the mTOR/S6 kinase pathway and ribosomal S6 phosphorylation. Second, the sensitivity to rapamycin in vivo under basal conditions is significantly different from that observed in cultured cells. Lastly, our study indicates that rapamycin resistance is not just an acquired attribute of malignant cells, but is an intrinsic characteristic of many tissues in vivo. Thus elucidation of the mechanism of rapamycin resistance in normal tissue may be relevant to carcinogenesis. We will continue to use the system of liver development versus regeneration to investigate this mechanism.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by United States Public Health Service (USPHS) Grants HD-24455 and HD-35831 (to P. Gruppuso), by a USPHS training grant (T32-GM-007601), and by the Rhode Island Hospital Department of Pediatrics Research Endowment Funds.


   ACKNOWLEDGMENTS
 
We thank Shu-Whei Tsai for assistance with the animal studies and Joan Boylan for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Sanders, Dept. of Pediatrics, Rhode Island Hospital, 593 Eddy St., Providence, RI 02903 (e-mail: Jennifer_Sanders{at}brown.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Anand P, Boylan JM, Ou Y, Gruppuso PA. Insulin signaling during perinatal liver development in the rat. Am J Physiol Endocrinol Metab 283: E844–E852, 2002.[Abstract/Free Full Text]

2. Bjornsti MA, Houghton PJ. The TOR pathway: a target for cancer therapy. Nat Rev Cancer 4: 335–348, 2004.[CrossRef][Web of Science][Medline]

3. Boylan JM, Anand P, Gruppuso PA. Ribosomal protein S6 phosphorylation and function during late gestation liver development in the rat. J Biol Chem 276: 44457–44463, 2001.[Abstract/Free Full Text]

4. Boylan JM, Gruppuso PA. Uncoupling of hepatic, epidermal growth factor-mediated mitogen-activated protein kinase activation in the fetal rat. J Biol Chem 273: 3784–3790, 1998.[Abstract/Free Full Text]

5. Breslin EM, White PC, Shore AM, Clement M, Brennan P. LY294002 and rapamycin co-operate to inhibit T-cell proliferation. Br J Pharmacol 144: 791–800, 2005.[CrossRef][Web of Science][Medline]

6. Chou MM, Masuda-Robens JM, Gupta ML. Cdc42 promotes G1 progression through p70 S6 kinase-mediated induction of cyclin E expression. J Biol Chem 278: 35241–35247, 2003.[Abstract/Free Full Text]

7. De Benedetti A, Harris AL. eIF4E expression in tumors: its possible role in progression of malignancies. Int J Biochem Cell Biol 31: 59–72, 1999.[CrossRef][Web of Science][Medline]

8. Dilling MB, Germain GS, Dudkin L, Jayaraman AL, Zhang X, Harwood FC, Houghton PJ. 4E-binding proteins, the suppressors of eukaryotic initiation factor 4E, are down-regulated in cells with acquired or intrinsic resistance to rapamycin. J Biol Chem 277: 13907–13917, 2002.[Abstract/Free Full Text]

9. Dufner A, Thomas G. Ribosomal S6 kinase signaling and the control of translation. Exp Cell Res 253: 100–109, 1999.[CrossRef][Web of Science][Medline]

10. Fingar DC, Blenis J. Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23: 3151–3171, 2004.[CrossRef][Web of Science][Medline]

11. Gaben AM, Saucier C, Bedin M, Barbu V, Mester J. Rapamycin inhibits cdk4 activation, p 21(WAF1/CIP1) expression and G1-phase progression in transformed mouse fibroblasts. Int J Cancer 108: 200–206, 2004.[CrossRef][Web of Science][Medline]

12. Gera JF, Mellinghoff IK, Shi Y, Rettig MB, Tran C, Hsu JH, Sawyers CL, Lichtenstein AK. AKT activity determines sensitivity to mammalian target of rapamycin (mTOR) inhibitors by regulating cyclin D1 and c-myc expression. J Biol Chem 279: 2737–2746, 2004.[Abstract/Free Full Text]

13. Hashemolhosseini S, Nagamine Y, Morley SJ, Desrivieres S, Mercep L, Ferrari S. Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability. J Biol Chem 273: 14424–14429, 1998.[Abstract/Free Full Text]

14. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 18: 1926–1945, 2004.[Abstract/Free Full Text]

15. Hosoi H, Dilling MB, Liu LN, Danks MK, Shikata T, Sekulic A, Abraham RT, Lawrence JC Jr, Houghton PJ. Studies on the mechanism of resistance to rapamycin in human cancer cells. Mol Pharmacol 54: 815–824, 1998.[Abstract/Free Full Text]

16. Huang S, Houghton PJ. Mechanisms of resistance to rapamycins. Drug Resist Updat 4: 378–391, 2001.[CrossRef][Web of Science][Medline]

17. Inoki K, Ouyang H, Li Y, Guan KL. Signaling by target of rapamycin proteins in cell growth control. Microbiol Mol Biol Rev 69: 79–100, 2005.[Abstract/Free Full Text]

18. Jefferies HB, Fumagalli S, Dennis PB, Reinhard C, Pearson RB, Thomas G. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. EMBO J 16: 3693–3704, 1997.[CrossRef][Web of Science][Medline]

19. Jiang YP, Ballou LM, Lin RZ. Rapamycin-insensitive regulation of 4e-BP1 in regenerating rat liver. J Biol Chem 276: 10943–10951, 2001.[Abstract/Free Full Text]

20. Kurmasheva RT, Huang S, Houghton PJ. Predicted mechanisms of resistance to mTOR inhibitors. Br J Cancer 95: 955–960, 2006.[CrossRef][Web of Science][Medline]

21. Law BK. Rapamycin: an anti-cancer immunosuppressant? Crit Rev Oncol Hematol 56: 47–60, 2005.[Web of Science][Medline]

22. Luo Y, Marx SO, Kiyokawa H, Koff A, Massague J, Marks AR. Rapamycin resistance tied to defective regulation of p27Kip1. Mol Cell Biol 16: 6744–6751, 1996.[Abstract]

23. Marks AR. Rapamycin: signaling in vascular smooth muscle. Transplant Proc 35: 231S–233S, 2003.[CrossRef][Web of Science][Medline]

24. Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY, Nakayama K. Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85: 707–720, 1996.[CrossRef][Web of Science][Medline]

25. Pende M, Um SH, Mieulet V, Sticker M, Goss VL, Mestan J, Mueller M, Fumagalli S, Kozma SC, Thomas G. S6K1(–/–)/S6K2(–/–) mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway. Mol Cell Biol 24: 3112–3124, 2004.[Abstract/Free Full Text]

26. Ruvinsky I, Sharon N, Lerer T, Cohen H, Stolovich-Rain M, Nir T, Dor Y, Zisman P, Meyuhas O. Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev 19: 2199–2211, 2005.[Abstract/Free Full Text]

27. Sehgal SN. Rapamune (RAPA, rapamycin, sirolimus): mechanism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression. Clin Biochem 31: 335–340, 1998.[CrossRef][Web of Science][Medline]

28. Sehgal SN. Sirolimus: its discovery, biological properties, and mechanism of action. Transplant Proc 35: 7S–14S, 2003.[CrossRef][Web of Science][Medline]

29. Shapira M, Kakiashvili E, Rosenberg T, Hershko DD. The mTOR inhibitor rapamycin down-regulates the expression of the ubiquitin ligase subunit Skp2 in breast cancer cells. Breast Cancer Res 8: R46, 2006.[CrossRef][Medline]

30. Singha UK, Jiang Y, Yu S, Luo M, Lu Y, Zhang J, Xiao G. Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3–E1 cells and primary mouse bone marrow stromal cells. J Cell Biochem 103: 434–446, 2008.[CrossRef][Web of Science][Medline]

31. Steer CJ. Liver regeneration. FASEB J 9: 1396–1409, 1995.[Web of Science][Medline]

32. Sun SY, Rosenberg LM, Wang X, Zhou Z, Yue P, Fu H, Khuri FR. Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 65: 7052–7058, 2005.[Abstract/Free Full Text]

33. Vaysberg M, Balatoni CE, Nepomuceno RR, Krams SM, Martinez OM. Rapamycin inhibits proliferation of Epstein-Barr virus-positive B-cell lymphomas through modulation of cell-cycle protein expression. Transplantation 83: 1114–1121, 2007.[CrossRef][Medline]

34. Vega F, Medeiros LJ, Leventaki V, Atwell C, Cho-Vega JH, Tian L, Claret FX, Rassidakis GZ. Activation of mammalian target of rapamycin signaling pathway contributes to tumor cell survival in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma. Cancer Res 66: 6589–6597, 2006.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/2/C406    most recent
00450.2007v1
Right arrow Alert me when this article is cited
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
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sanders, J. A.
Right arrow Articles by Gruppuso, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sanders, J. A.
Right arrow Articles by Gruppuso, P. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.