help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1213
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
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 Grimberg, A.
Right arrow Articles by El-Deiry, W. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grimberg, A.
Right arrow Articles by El-Deiry, W. S.
Related Collections
Right arrow Metabolism
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3568-3574
Copyright © 2005 by The Endocrine Society

p53-Dependent and p53-Independent Induction of Insulin-Like Growth Factor Binding Protein-3 by Deoxyribonucleic Acid Damage and Hypoxia

Adda Grimberg, Carrie M. Coleman, Timothy F. Burns, Bruce P. Himelstein, Cameron J. Koch, Pinchas Cohen and Wafik S. El-Deiry

Department of Pediatric Endocrinology (A.G., C.M.C.), The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318; Department of Medicine (T.F.B., W.S.E.-D.), University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6140; Department of Radiation Oncology (C.J.K.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4385; Department of Pediatric Oncology (B.P.H.), Children’s Hospital of Wisconsin, Milwaukee, Wisconsin 53201-1997; and Department of Pediatric Endocrinology (P.C.), University of California, Los Angeles, Los Angeles, California 90095

Address all correspondence and requests for reprints to: Adda Grimberg, M.D., Assistant Professor, Division of Pediatric Endocrinology, The Children’s Hospital of Philadelphia, Abramson Research Center, Room 802, 3615 Civic Center Boulevard, Philadelphia, Pennsylvania 19104-4318. E-mail: grimberg{at}e-mail.chop.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF binding protein (IGFBP)-3, the principal carrier of IGFs in the circulation, contributes to both endocrine and autocrine/paracrine growth control; it can be induced by GH, cytokines, retinoic acid, and tumor suppressors. Induction of IGFBP-3 by the tumor suppressor p53 has been shown in various models that directly manipulate p53 activity. However, the physiologic settings under which this induction occurs have not been established. DNA damage and hypoxia are two important physiologic activators of p53. We have demonstrated for the first time that IGFBP-3 is an in vivo target of p53 in response to ionizing radiation. This effect was tissue specific. Furthermore, we demonstrated that genotoxic drugs could increase IGFBP-3 protein levels and secretion in tumor cell lines in a p53-independent manner. Finally, we have established that IGFBP-3 induction under hypoxic conditions is independent of p53 in tumor cell lines derived form multiple tissue types. Thus, IGFBP-3 is induced by physiologic conditions that also induce p53, although p53 is not always required. Because IGFBP-3 can inhibit growth and induce apoptosis in IGF-dependent and IGF-independent manners, its induction by DNA damage and hypoxia suggest IGFBP-3 plays a role in the physiologic protection against aberrant cell growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MUTATIONS IN P53 are the most frequently occurring genetic lesions among human cancers, and many of the cancers that do not harbor p53 mutations have mutations in regulators of p53 function (1, 2). p53 normally resides in low concentrations in a latent form but is activated via protein accumulation, posttranslational modifications (3), and subcellular translocation (4, 5) to induce cell cycle arrest or apoptosis, thereby preventing the propagation of damaged cells. The stimuli for p53 activation include DNA damage, chromosomal defects, telomere shortening, cellular hypoxia, and oncogene overexpression, and their diversity contributes to the importance of p53 as a tumor suppressor (6). Once activated, p53 causes cell cycle arrest or apoptosis primarily through sequence-specific transcriptional transactivation of target genes (7) and, to a lesser extent, transcriptional repression, protein-protein interactions, and direct mitochondrial activities (8).

IGF binding protein (IGFBP)-3 is one of the genes transcriptionally activated by p53 (9, 10). Originally identified as the principal carrier of IGFs in the circulation and as such, an important component of the GH-IGF axis, IGFBP-3 also plays a role in autocrine and paracrine growth control and apoptosis (11). Multiple p53 mutants with selective loss of the ability to transactivate IGFBP-3 and Bcl-2-associated X protein could not induce apoptosis, whereas mutants without p21WAF1/CIP1 transactivation could not induce cell cycle arrest (12, 13, 14). We have previously demonstrated that IGFBP-3 mediates p53-induced apoptosis during serum starvation using two contrasting neoplastic cell models (15). p53-null prostate cancer cells were stably transfected with a doxycycline-inducible p53 construct to turn p53 on, and lung cancer cells containing fully functional wild-type p53 were stably transfected with human papillomavirus E6, which targets p53 for ubiquitin-mediated degradation (16, 17), to turn p53 off. Whereas these two models enabled us to directly manipulate p53 levels to study the effects on IGFBP-3 and apoptosis, they are artificial constructs. The question remains, what is the physiologic context of IGFBP-3 induction by p53? If IGFBP-3 were a physiologic target of p53, then it should be induced by conditions that physiologically induce p53.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and treatments

Healthy 6- to 7-wk-old female p53–/– and p53+/+ mice were obtained from Jackson Laboratories (Bar Harbor, ME). Two animals in each experimental group received a single ip injection of either 0.5 mg dexamethasone (Elkins-Sinn Inc., Cherry Hill, NJ) or sterile 1x PBS. Two animals of each genotype received 5 Gy total body irradiation from a 137Cesium {gamma}-source at a dose rate of 1.532 Gy/min. The mice were killed at 6 and 24 h using an approved protocol (University of Pennsylvania Institutional Animal Care and Use Committee), which followed recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Tissues were harvested, snap frozen in liquid nitrogen, and kept at –80 C until used for the Taqman RT-PCR experiments.

Taqman real-time quantitative RT-PCR

Total RNA was isolated from radiation-sensitive tissues as previously described (18), and 1 µg was used for reverse transcription (RT) and amplification using TaqMan reverse transcription reagents according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA). Parallel samples were similarly treated but without adding reverse transcriptase (–RT) to provide a control for genomic contamination. Taqman RT-PCR assay was conducted according to the manufacturer’s specifications (Applied Biosystems) and as previously reported for other p53 target genes (19). A master mix of TaqMan reagents was prepared, and 10 ng of each RT (–RT) sample were used in the Taqman PCR. Each tube contained a probe and primer set for both IGFBP-3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control; the IGFBP-3 probe was labeled with FAM (6-carboxyfluorescin), whereas that for GAPDH was labeled with VIC (Applied Biosystems). Using Primer Express version 1.0 (Applied Biosystems), the following primer and probe sequences were designed to span IGFBP-3 intron/exon boundaries to prevent amplification of genomic DNA: 5'-CAAAGCACAGACACCCAGAACTT-3', 5'-TGCGGCAGGGACCGT-3', and probe 6FAM-TCCTCCGAGTCTAAGCGGGAGACAGAA. GAPDH primers and VIC-labeled probe were obtained from Applied Biosystems.

RT-PCRs were carried out in 96-well plates using the ABI PRISM 7700 sequence detection system (Applied Biosystems). Each sample was analyzed in quadruplicate, and Taqman master mix with no template served as negative control. Because the increase in fluorescence was proportional to the concentration of template in the PCR, the standard curve method was used to quantitate the amount of IGFBP-3 in each reaction according to manufacturer’s protocol (Applied Biosystems). Any contribution of genomic contamination to the threshold cycle value for a sample was ignored because the threshold cycle values of the –RT reactions were at least 10 cycles less than for the corresponding samples in which reverse transcriptase was added. All IGFBP-3 measurements were normalized to GAPDH concentrations to control for any loading differences.

Cell culture treatment with genotoxic drugs

As previously reported (15, 20), two sister cell lines created from the p53+/+ human lung carcinoma cell line (H460) differ in their p53 content; H460 cells stably transfected with a plasmid containing the gene for E6 (H460-E6) contain far less p53 than H460 cells stably transfected with the empty plasmid as control (H460-neo). E6, a protein produced by tumor-associated human papillomaviruses (HPV types 16 and 18), binds p53 and targets it for ubiquitin-dependent degradation (16, 17). E6 has more recently been shown to also inhibit the acetylation, and hence activation, of p53 (21). H460 cells were maintained at all times in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) with 500 µg/ml G418 (Mediatech, Inc., Herndon, VA) for continued transfectant selection pressure. p53–/– PC-3 cells (American Type Culture Collection, Manassas, VA) were maintained in F-12K nutrient mixture (Kaighn’s modification) (Life Technologies).

Cells were plated in equal cell densities onto 6-well plates. After adhering overnight in complete medium, duplicate wells were changed to serum-free medium with or without a genotoxic drug at concentrations previously shown to induce wild-type p53 expression (20). Etoposide (Bristol Laboratories, Princeton, NJ) was added for a final concentration of 2.4 µM. Adriamycin (Pharmacia & Upjohn Co., Kalamazoo, MI) was added at 0.2 µg/ml, as published, or 0.8 µg/ml to test the hypothesis that higher concentrations may be needed in p53-null cells. Conditioned medium was harvested at the times shown, and IGFBP-3 immunoblot was performed by established techniques (22) using an affinity purified, polyclonal goat antihuman IGFBP-3 antibody (Diagnostic Systems Laboratories, Webster, TX). Densitometry analysis was performed (Alpha imager 2000 4.0, Alpha Innotech, San Leandro, CA) to quantify the relative amounts of IGFBP-3 protein in the conditioned media.

To control for possible loading differences, aliquots of each conditioned medium were analyzed by the modified Lowry technique for total protein content (23). Serial dilutions of a standard protein solution of 0.1 mg/ml BSA (Sigma, St. Louis, MO) were used to generate the standard curve. For each experimental conditioned medium, one aliquot was diluted 20-fold and another 40-fold, to get readings within the range of the BSA standards. Then 0.1ml of a 5-ml carbonate solution/0.1ml copper sulfate solution mixture was added to each cuvette, and 1 min later, 0.1 ml Folin reagent (diluted 1:1 with water) was also added. After 30 min, the spectrophometric absorbances were read at 710 nm ({lambda} 2 UV/Vis spectrophotometer, PerkinElmer Life and Analytical Sciences, Inc., Boston, MA) and converted into protein concentrations using the standard curve. The protein concentrations of the two aliquots for each condition were averaged and used to normalize the quantities of IGFBP-3 protein as determined by the densitometry readings.

Hypoxia

Hypoxic conditions for thin film cell cultures were achieved by the gas exchange procedure (24) using an MS2200V manifold (Animas Corp., Philadelphia, PA). Cells were plated at 2 x106 cells per 4 ml/dish on 60-mm Permanox dishes that had been previously degassed for 48 h (25). After 24 h in standard medium, cells were changed to thin-layer medium supplemented with 1 M HEPES and 75 mg glucose to prevent acidosis and placed in the MS2200V Manifold chambers (24). Sequential gas exchanges to stepwise replace the chamber’s air content with nitrogen mixed with 5% CO2 were undertaken; serial exchanges were required to avoid large vacuum differentials that could impair cell adherence and viability. Once the desired hypoxic condition was created, the chambers were incubated in a reciprocal shaker for the experimental duration to maximize equilibration. Afterward, the cells were harvested, and an aliquot of the medium was examined for pH and glucose content. Per established techniques, total RNA was isolated from the cells, and IGFBP-3 mRNA content was analyzed by Northern blotting. RPL-32 mRNA was used to control for loading differences in the Northern blots.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Irradiation

We examined whether IGFBP-3 would be induced by p53 in multiple tissues (thymus, spleen, small intestine) that have been previously shown to undergo p53-dependent apoptosis after treatment with ionizing radiation. As shown in Fig. 1Go, IGFBP-3 mRNA increased in the thymus of p53+/+ mice by almost 8-fold at 6 h and 22-fold at 24 h after total body irradiation. Similar induction was not observed in the p53–/– mice. Dexamethasone treatment, which induces apoptosis by a p53-independent pathway, served as a positive control in the experimental design. In similar experiments (data not shown), irradiation led to a p53-dependent doubling of IGFBP-3 mRNA in the small intestine and no change in IGFBP-3 mRNA in the spleen.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 1. IGFBP-3 mRNA expression in the thymus after irradiation or dexamethasone (Dex) treatment. Pairs of p53+/+ and p53–/– mice were treated with 5 Gy total body irradiation or a single ip injection of either 0.5 mg dexamethasone or sterile 1x PBS and killed 6 or 24 h later. Radiation-sensitive tissues were harvested (thymus shown here), total RNA isolated, and RT-PCR performed to quantify IGFBP-3 mRNA levels, normalized to GAPDH. RT-PCR results presented as fold increase relative to untreated p53+/+ mice (mean of quadruplicate replications ± SD).

 
Genotoxic drugs

We then examined whether other DNA damaging agents could induce IGFBP-3 in a p53-dependent manner. IGFBP-3 Western immunoblots were performed on the conditioned media of H460 cells after 3 h of serum starvation with or without exposure to etoposide (Fig. 2Go). Similar to prior report (15), H460 cells stably transfected with E6 (H460-E6) have less p53 and also less IGFBP-3 than H460 cells transfected with empty plasmid (H460-neo). Nonetheless, etoposide treatment increased IGFBP-3 secretion by both H460-E6 and H460-neo cells. To further assess the p53-independence of IGFBP-3 induction by genotoxic drugs, p53-null PC-3 cells were exposed to serum starvation and adriamycin for 6 h. As shown in Fig. 3Go, adriamycin more than doubled the IGFBP-3 secretion by PC-3 cells at 0.2 µg/ml, the concentration of adriamycin previously reported in studies of p53+/+ cell lines (20).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 2. IGFBP-3 secretion by H460 cells after 3 h treatment with etoposide A, Western immunoblot. B, Densitometry analysis. p53+/+ H460 cells had been stably transfected with E6 or empty plasmid as control (neo); E6 leads to p53 degradation and at 3 h significantly reduced IGFBP-3 secretion (15 ). Conditioned media were collected after 3 h in serum-free medium with or without etoposide. IGFBP-3 Western immunoblot was performed as was densitometry analysis. An aliquot of each sample underwent total protein quantification by the modified Lowry technique to control for loading differences. Densitometry results, normalized to total protein, are presented as percent relative to untreated H460-E6 cells. Each bar represents mean ± SD of triplicate replications.

 


View larger version (29K):
[in this window]
[in a new window]
  		FIG. 3. IGFBP-3 secretion by p53–/– PC-3 cells after 6 h treatment with different concentrations of adriamycin. A, Western immunoblot. B, Densitometry analysis. Conditioned media were collected after p53–/– PC-3 cells were treated with serum-free medium with or without adriamycin at different concentrations. IGFBP-3 Western immunoblot and densitometry analysis were performed. An aliquot of each sample underwent total protein quantification by the modified Lowry technique to control for loading differences. Densitometry results, normalized to total protein, are presented as percent relative to untreated PC-3 cells. Each bar represents mean ± SD of duplicate replications.

 
Hypoxia

Thin film cell cultures were subjected to hypoxia by the gas exchange procedure. Hypoxic conditions induce a metabolic switch to glycolysis, which results in accelerated glucose deprivation and cellular acidosis (26). Distinguishing the effects of hypoxia from those of the resultant acidosis becomes an important feature of the experimental design. To this end, the thin layer medium was supplemented with 1 M HEPES and 75 mg glucose, and the adequacy of this approach in preventing acidosis was confirmed (Fig. 4Go). Figure 5Go shows the time course and dose response of IGFBP-3 induction by hypoxia in p53+/+ H460 cells. IGFBP-3 mRNA increased progressively with the duration of exposure to oxygen partial pressure of 1%, and at 24 h, IGFBP-3 mRNA was significantly greater in cells kept in oxygen partial pressures of 1% or less. A similar dose response of IGFBP-3 mRNA was seen in p53–/– PC-3 cells (Fig. 6Go). In fact, 24 h of hypoxia at oxygen partial pressure of 1% induced IGFBP-3 mRNA across multiple neoplastic cell lines irrespective of their p53 status (Fig. 7Go); these included cell lines derived from osteogenic sarcoma (Saos-2), breast adenocarcinoma (MCF7), cervical carcinoma (HeLa), prostate carcinoma (PC-3 and DU145), and lung large cell carcinoma (H460-E6 and H460-neo).



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 4. pH of conditioned medium after 24 h at different O2 partial pressures. PC-3 cells are represented by open black squares/solid line, H460 cells by closed gray boxes/dashed line. Thin film cell cultures were subjected to increasing degrees of hypoxia by the gas exchange procedure using an MS2200V manifold and incubated in a reciprocal shaker for 24 h. Afterward, the cells were harvested for Northern blot analysis (Figs. 5BGo and 6Go), and an aliquot of the medium was examined for pH and glucose content. Before any changes can be attributed to hypoxia, it is important to ensure that cellular acidosis, which frequently results from hypoxia, is prevented.

 


View larger version (79K):
[in this window]
[in a new window]
  		FIG. 5. Time course (A) and dose response (B) of IGFBP-3 mRNA levels in p53+/+ H460 cells treated with hypoxia. Thin film cultures of H460 cells were subjected to 1% oxygen partial pressure for different durations (A) or 24 h of progressively decreasing oxygen partial pressures (B). Cells were harvested, total RNA isolated, and IGFBP-3 mRNA analyzed by Northern blotting. RPL-32 is shown as loading control.

 


View larger version (51K):
[in this window]
[in a new window]
  		FIG. 6. Dose response of IGFBP-3 mRNA levels in p53-null PC-3 cells after 24 h of varying degrees of hypoxia. The experiment of Fig. 5BGo was performed with PC-3 cells. Northern blotting was used to evaluate IGFBP-3 mRNA levels, and RPL-32 served as loading control.

 


View larger version (20K):
[in this window]
[in a new window]
  		FIG. 7. Northern blots of IGFBP-3 mRNA in multiple human cancer cell lines. Using the thin film cell culture technique, each cell type was exposed to 24 h of hypoxia (H; P02 = 1%) or normoxia for control (C). After the 24-h incubation in a reciprocal shaker, cells were harvested, total RNA isolated, and IGFBP-3 mRNA evaluated by Northern blot analysis. The p53 status of each cell type is indicated above its doublet.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In summary, irradiation, genotoxic drugs, and hypoxia, three conditions that normally induce p53, all induced IGFBP-3, although p53 was not always required. Irradiation significantly induced IGFBP-3 in the thymus and, to a lesser extent, the small intestine, in a p53-dependent and tissue-specific manner. p53 is a determinant of tissue-specific radiosensitivity, and the most radiosensitive tissues (hematopoietic and lymphoid tissues, spermatogonia, small intestine, hair follicles, and embryos) show the greatest differences in radiosensitivity between p53+/+ and p53–/– mice (27). Irradiation causes DNA damage, which is detected by sensor proteins and transduced through ataxia telangiectasia mutated gene (ATM)/ataxia-telangiectasia and rad3-related (ATR) to effector kinases; ATM, both directly and through its targets, leads to p53 stabilization and activation, which ultimately contributes to the cell’s fate of DNA repair, cell cycle arrest, or apoptosis (28). In situ hybridization experiments of irradiated mouse thymus, spleen, and various intestinal segments showed tissue-specific induction patterns of the p53 targets KILLER/DR5, Bid, Noxa, and Puma, that correlated with caspase-3 activation, a marker of apoptosis. Caspase-3 was not activated in irradiated p53–/– mice or the livers of p53+/+ mice; p21WAF1, the principal mediator of p53-induced cell cycle arrest, was the major p53 target induced in the liver (29). Thus, it is not surprising for IGFBP-3 induction by irradiation to be p53 dependent and tissue specific. The magnitude of IGFBP-3 induction in the thymus was on par with that seen, under similar experimental conditions, for the p53 targets KILLER/DR5, Bcl-2-associated X protein, E128/PIG8, and Fas/APO1 (19).

p53 is induced by DNA damage, whether it is caused by irradiation or genotoxic drugs. In fact, most clinically used chemotherapeutic agents kill p53+/+ tumors more effectively than tumors with mutant p53 (30), although their use is not limited to p53+/+ tumors. Cellular responses to DNA damage are specific and relate to the type and extent of DNA damage, degree of p53 induction, and cell type (6). Etoposide causes DNA strand breakage, via interactions with DNA topoisomerase II or formation of free radicals, whereas adriamycin intercalates nucleotide bases, thereby inhibiting DNA replication and DNA and RNA polymerases (31). Etoposide increased IGFBP-3 secretion by H460 lung cancer cells with or without E6, and adriamycin increased IGFBP-3 in the p53-null prostate cancer cells, PC-3. The effects were rapid (evident within 3–6 h), p53-independent, and specific to the combination of cell type and damaging agent.

How can genotoxic drugs induce IGFBP-3 in the absence of p53? One possibility is p73, a p53 family member that is rarely mutated (and frequently increased) in cancer. p73 and the third family member, p63, were required for adriamycin-induced apoptosis despite normal p53 status (32). Recently p73 was found to be induced in SW480 colon carcinoma cells by multiple chemotherapeutic agents, including etoposide and adriamycin and in turn led to apoptosis, even though these tumor cells lacked functional p53 (33). Created by different promoters and alternative splicing, isoforms of p73 and p63 that contain the N-terminal transactivation domain can activate p53 target genes and cause apoptosis; the isoforms lacking this domain function as dominant inhibitors of p53 (34). Thus, p53-independent induction of IGFBP-3 by genotoxic drugs may be mediated by p73 or perhaps by p63 or other p53-unrelated signals.

IGFBP-3 was significantly induced by hypoxia at oxygen partial pressures of 1% or less. Acidosis, due to cellular hypoxia, was prevented (Fig. 4Go). It is important to distinguish the effects of hypoxia itself vs. those of the ensuing acidosis. This technical question is particularly relevant to studies of IGFBP-3 biology because multiple IGFBP-3 regulators are themselves regulated by acidosis. For example, cathepsins, lysosomal enzymes implicated in malignant processes, are acid activated (35), whereas prostate-specific antigen is functional at neutral pH (36). Cathepsins and prostate-specific antigen appear to be secreted into seminal plasma, but their role there is not yet known. They can both function as IGFBP-3 proteases, and increased IGFBP-3 proteolysis has been found in prostate carcinogenesis (35, 37).

Hypoxia inhibits cell growth yet can lead to a more malignant phenotype in tumors by inducing genes whose products are involved in angiogenesis and energy metabolism; IGFBP-3 was one of multiple hypoxia-induced genes identified by DNA array analysis (38). The hypoxic response is primarily mediated by the transcription factor hypoxia- inducible factor (HIF-1), which is frequently overexpressed in human cancers (39, 40). Hypoxia was shown to induce IGFBP-3 mRNA in wild-type embryonic stem cells, whereas stem cells homozygous for a targeted loss of the HIF-1{alpha} subunit had undetectable levels of IGFBP-3 mRNA (41). HIF-1{alpha} was required for induction of p53 by anoxia/hypoglycemia (42); HIF-1{alpha} overexpression protected p53 from mouse double minute-2-homolog (Mdm2) mediated proteasomal degradation and inhibited nuclear export of p53 by mouse double minute-2 homolog (Mdm2) (43). It is tempting to assume that hypoxic induction of IGFBP-3 is mediated by increased IGFBP-3 transcription as a p53 target gene. However, our results clearly indicate that hypoxia can increase IGFBP-3 mRNA levels, even in the absence of p53. A recent paper (44) demonstrated uncoupling of HIF-1{alpha} and p53 expression during the cellular response to hypoxia; p53 induction by hypoxia occurred not at reduced oxygen tensions alone but when the ensuing acidosis and nutrient deprivation were allowed as well. Hypoxia-induced growth arrest was abolished by deletion of HIF-1{alpha}, also in a p53-independent manner (45). More experiments are needed to determine whether this p53-independent, hypoxia- and HIF1{alpha}-induced growth arrest is due to IGFBP-3 activity.

IGFBP-3 is not the only member of the IGF axis regulated by hypoxia. HIF-1 activates transcription of numerous target genes through DNA sequence-specific binding in a hypoxia response element (HRE) that can be located in the 5' or 3' flanking regions or in the intronic sequences of the target gene (38, 39). Giudice and colleagues (46, 47) described a potent response of IGFBP-1 to hypoxia via canonic HREs and proposed that this may contribute to the intrauterine growth retardation of hypoxic fetuses. Insulin, IGF-I, and IGF-II were shown to induce expression of HIF-1{alpha}, which in turn was required for hypoxic induction of IGF-II, IGFBP-2, and IGFBP-3 but not IGFBP-4 (41). This may represent a negative feedback autocrine growth factor loop. IGFBP-1, -2, and -3 all contain two to three canonic HREs at various locations of their genomic sequence (data not shown) and may have coevolved to maintain their role as regulatory molecules for hypoxia-induced proliferation.

IGFBP-3 is also not the only member of the IGF axis regulated by p53. Like HIF-1, p53 activates transcription of numerous target genes through DNA sequence-specific binding (48). For IGFBP-3, the p53 binding sites are located in its first two introns (9) and promoter (10, 49). In addition, p53 can repress transcription of another set of target genes. p53 was shown to transcriptionally repress, by inhibiting DNA binding of the TATA-box-binding protein subunit of the transcription factor TFIID, to the initiator region of the type 1 IGF receptor gene (50) and the third promoter (P3) of the IGF-II gene (51). By inducing IGFBP-3 and repressing the IGF receptor and IGF-II, p53 effectively inhibits the cell survival and proliferation signaling of the IGF axis. For example, tetracycline-inducible p53 expression in an osteosarcoma cell line reduced IGF-I-induced tyrosine phosphorylation of the IGF receptor and insulin receptor substrate-1 (52), and overexpression of wild-type but not mutant p53 in murine hemopoietic cells decreased the number of IGF receptors and increased cellular sensitivity to apoptosis caused by IL-3 withdrawal (53).

In conclusion, we have shown that IGFBP-3 is induced by physiologic conditions that also induce p53, although p53 is not always required. IGFBP-3 induction by DNA damage and hypoxia suggest IGFBP-3 plays a role in the physiologic protection against aberrant cell growth. This may be accomplished through two principal mechanisms (11). First, because the IGF binding affinity for IGFBP-3 exceeds that for the type 1 IGF receptor, IGFBP-3 competitively inhibits IGF signaling. Current evidence supports a permissive, but not causal, role for IGF-I in cancer development (54). Second, IGFBP-3 can inhibit growth and induce apoptosis in an IGF-independent fashion that involves IGFBP-3-specific association proteins, including putative IGFBP-3 cell surface receptors (55, 56) and retinoid X receptor in the nucleus (57). Accumulating evidence, from epidemiologic studies of cancer risk to in vivo tumor models to in vitro cell growth experiments, suggests IGFBP-3 contributes to cancer protection (58). Unlike classic tumor suppressors, IGFBP-3 knockout mice do not develop spontaneous tumors, and there is no identified IGFBP-3-related familial cancer syndrome. Thus, IGFBP-3 more likely serves as one of the multiple, low-penetrance tumor susceptibility and resistance genes that determine cancer incidence and therapeutic responsiveness (59).


   Footnotes
 
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant K08 DK064352 (to A.G.) and Lawson Wilkins Pediatric Endocrine Society Genentech Clinical Scholar Award (to A.G.).

First Published Online March 15, 2005

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HIF, hypoxia-inducible factor; HRE, hypoxia response element; IGFBP, IGF binding protein; RT, reverse transcription; –RT, samples treated similarly to RT but without adding reverse transcriptase.

Received June 30, 2004.

Accepted March 4, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hainaut P, Hollstein M 2000 p53 and human cancer: the first ten thousand mutations. Adv Cancer Res 77:81–137[Medline]
  2. Vousden KH, Prives C 2005 p53 and prognosis: new insights and further complexity. Cell 120:7–10[Medline]
  3. Brooks CL, Gu W 2003 Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr Opin Cell Biol 15:164–171[CrossRef][Medline]
  4. Fabbro M, Henderson BR 2003 Regulation of tumor suppressors by nuclear-cytoplasmic shuttling. Exp Cell Res 282:59–69[CrossRef][Medline]
  5. Gostissa M, Hofmann TG, Will H, Del Sal G 2003 Regulation of p53 functions: let’s meet at the nuclear bodies. Curr Opin Cell Biol 15:351–357[CrossRef][Medline]
  6. Oren M 2003 Decision making by p53: life, death and cancer. Cell Death Differ 10:431–442[CrossRef][Medline]
  7. Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW, Vogelstein B 1999 Identification and classification of p53-regulated genes. Proc Natl Acad Sci USA 96:14517–14522[Abstract/Free Full Text]
  8. Balint EE, Vousden KH 2001 Activation and activities of the p53 tumour suppressor protein. Br J Cancer 85:1813–1823[CrossRef][Medline]
  9. Buckbinder L, Talbott R, Velasco-Miguel S, Takenaka I, Faha B, Seizinger BR, Kley N 1995 Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature 377:646–649[CrossRef][Medline]
  10. Bourdon JC, Deguin-Chambon V, Lelong JC, Dessen P, May P, Debuire B, May E 1997 Further characterisation of the p53 responsive element-identification of new candidate genes for trans-activation by p53. Oncogene 14:85–94[CrossRef][Medline]
  11. Grimberg A, Cohen P 2000 The role of insulin-like growth factors and their binding proteins in growth control and carcinogenesis. J Cell Physiol 183:1–9[CrossRef][Medline]
  12. Friedlander P, Haupt Y, Prives C, Oren M 1996 A mutant p53 gene that discriminates between p53-responsive genes cannot induce apoptosis. Mol Cell Biol 16:4961–4971[Abstract]
  13. Ludwig RL, Bates S, Vousden KH 1996 Differential activation of target cellular promoters by p53 mutants with impaired apoptotic function. Mol Cell Biol 16:4952–4960[Abstract]
  14. Ryan KM, Vousden KH 1998 Characterization of structural p53 mutants which show selective defects in apoptosis but not cell cycle arrest. Mol Cell Biol 18:3692–3698[Abstract/Free Full Text]
  15. Grimberg A, Liu B, Bannerman P, El-Deiry WS, Cohen P 2002 IGFBP-3 mediates p53-induced apoptosis during serum starvation. Int J Oncol 21:327–335[Medline]
  16. Thomas M, Matlashewski G, Pim D, Banks L 1996 Induction of apoptosis by p53 is independent of its oligomeric state and can be abolished by HPV-18 E6 through ubiquitin mediated degradation. Oncogene 13:265–273[Medline]
  17. Mantovani F, Banks L 2001 The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene 20:7874–7887[CrossRef][Medline]
  18. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B 1993 WAF1, a potential mediator of p53 tumor suppression. Cell 75:817–825[CrossRef][Medline]
  19. Burns TF, Bernhard EJ, El-Deiry WS 2001 Tissue specific expression of p53 target genes suggests a key role for KILLER/DR5 in p53-dependent apoptosis in vivo. Oncogene 20:4601–4612[CrossRef][Medline]
  20. Wu GS, El-Deiry WS 1996 Apoptotic death of tumor cells correlates with chemosensitivity, independent of p53 or bcl-2. Clin Cancer Res 2:623–633[Abstract]
  21. Thomas MC, Chiang CM 2005 E6 Oncoprotein represses p53-dependent gene activation via inhibition of protein acetylation independently of inducing p53 degradation. Mol Cell 17:251–264[CrossRef][Medline]
  22. Coleman C, Grimberg A 2002 Alterations in the expression of insulin-like growth factors and their binding proteins in lung cancer. In: Driscoll B, ed. Methods in molecular medicine: lung cancer. Vol 1. Molecular pathology methods and reviews. Totawa, NJ: The Humana Press Inc.; 167–186
  23. Wahl R, Geissler W, Maasch HJ 1985 Comparison of classical Lowry, modified Lowry and a dye-binding assay for the estimation of protein in allergen extracts and influence of different parameters on the modified Lowry assay. Biol Chem Hoppe Seyler 366:979–984[Medline]
  24. Koch CJ 1984 A "thin-film" culturing technique allowing rapid gas-liquid equilibration (6 seconds) with no toxicity to mammalian cells. Radiat Res 97:434–442[Medline]
  25. Koch CJ, Kruuv J 1972 The release of oxygen from polystyrene Petri side dishes. Br J Radiol 45:787–788[Abstract/Free Full Text]
  26. Seagroves TN, Ryan HE, Lu H, Wouters BG, Knapp M, Thibault P, Laderoute K, Johnson RS 2001 Transcription factor HIF-1 is a necessary mediator of the Pasteur effect in mammalian cells. Mol Cell Biol 21:3436–3444[Abstract/Free Full Text]
  27. Gudkov AV, Komarova EA 2003 The role of p53 in determining sensitivity to radiotherapy. Nat Rev Cancer 3:117–129[CrossRef][Medline]
  28. Fei P, El-Deiry WS 2003 p53 and radiation responses. Oncogene 22:5774–5783[CrossRef][Medline]
  29. Fei P, Bernhard EJ, El-Deiry WS 2002 Tissue-specific induction of p53 targets in vivo. Cancer Res 62:7316–7327[Abstract/Free Full Text]
  30. El-Deiry WS 2003 The role of p53 in chemosensitivity and radiosensitivity. Oncogene 22:7486–7495[CrossRef][Medline]
  31. Perry, MC 2001 The chemotherapy source book. 3rd ed. Philadelphia: Lippincott, Williams and Wilkins
  32. Flores ER, Tsai KY, Crowley D, Sengupta S, Yang A, McKeon F, Jacks T 2002 p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature 416:560–564[CrossRef][Medline]
  33. Irwin MS, Kondo K, Marin MC, Cheng LS, Hahn WC, Kaelin Jr WG 2003 Chemosensitivity linked to p73 function. Cancer Cell 3:403–410[CrossRef][Medline]
  34. Benard J, Douc-Rasy S, Ahomadegbe JC 2003 TP53 family members and human cancers. Hum Mutat 21:182–191[CrossRef][Medline]
  35. Nunn SE, Peehl DM, Cohen P 1997 Acid-activated insulin-like growth factor binding protein protease activity of Cathepsin D in normal and malignant prostatic epithelial cells and seminal plasma. J Cell Physiol 171:196–204[CrossRef][Medline]
  36. Cohen P, Graves HC, Peehl DM, Kamarei M, Giudice LC, Rosenfeld RG 1992 Prostate specific antigen (PSA) is an IGF binding protein (IGFBP-3) protease found in seminal plasma. J Clin Endocrinol Metab 75:1046–1053[Abstract]
  37. Grimberg A, Rajah R, Zhao H, Cohen P 1998 The prostatic IGF system: new levels of complexity. In: Takano K, Hizuka N, Takahashi SI, eds. Molecular mechanisms to regulate the activities of insulin-like growth factors. Amsterdam: Elsevier Science B.V.; 205–213
  38. Koong AC, Denko NC, Hudson KM, Schindler C, Swiersz L, Koch C, Evans S, Ibrahim H, Le QT, Terris DJ, Giaccia AJ 2000 Candidate genes for the hypoxic tumor phenotype. Cancer Res 60:883–887[Abstract/Free Full Text]
  39. Semenza GL 2000 HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 88:1474–1480[Abstract/Free Full Text]
  40. Huang LE, Bunn HF 2003 Hypoxia-inducible factor and its biomedical relevance. J Biol Chem 278:19575–19578[Free Full Text]
  41. Feldser D, Agani F, Iyer NV, Pak B, Ferreira G, Semenza GL 1999 Reciprocal positive regulation of hypoxia-inducible factor 1{alpha} and insulin-like growth factor 2. Cancer Res 59:3915–3918[Abstract/Free Full Text]
  42. Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell P, Koch CJ, Ratcliffe P, Moons L, Jain RK, Collen D, Keshert E, Keshet E 1998 Role of HIF-1{alpha} in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394:485–490[CrossRef][Medline]
  43. Chen D, Li M, Luo J, Gu W 2003 Direct interactions between HIF-1{alpha} and Mdm2 modulate p53 function. J Biol Chem 278:13595–13598[Abstract/Free Full Text]
  44. Pan Y, Oprysko PR, Asham AM, Koch CJ, Simon MC 2004 p53 cannot be induced by hypoxia alone but responds to the hypoxic microenvironment. Oncogene 23:4975–4983[CrossRef][Medline]
  45. Goda N, Ryan HE, Khadivi B, McNulty W, Rickert RC, Johnson RS 2003 Hypoxia-inducible factor 1{alpha} is essential for cell cycle arrest during hypoxia. Mol Cell Biol 23:359–369[Abstract/Free Full Text]
  46. Tazuke SI, Mazure NM, Sugawara J, Carland G, Faessen GH, Suen LF, Irwin JC, Powell DR, Giaccia AJ, Giudice LC 1998 Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: a possible model for IGFBP-1 expression in fetal hypoxia. Proc Natl Acad Sci USA 95:10188–10193[Abstract/Free Full Text]
  47. Popovici RM, Lu M, Bhatia S, Faessen GH, Giaccia AJ, Giudice LC 2001 Hypoxia regulates insulin-like growth factor-binding protein 1 in human fetal hepatocytes in primary culture: suggestive molecular mechanisms for in utero fetal growth restriction caused by uteroplacental insufficiency. J Clin Endocrinol Metab 86:2653–2659[Abstract/Free Full Text]
  48. El-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B 1992 Definition of a consensus binding site for p53. Nat Genet 1:45–49[CrossRef][Medline]
  49. Hanafusa T, Shinji T, Shiraha H, Nouso K, Iwasaki Y, Yumoto E, Ono T, Koide N 2005 Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation. BMC Cancer 5:9[CrossRef][Medline]
  50. Werner H, Karnieli E, Rauscher FJ, LeRoith D 1996 Wild-type and mutant p53 differentially regulate transcription of the insulin-like growth factor I receptor gene. Proc Natl Acad Sci USA 93:8318–8323[Abstract/Free Full Text]
  51. Zhang L, Zhan Q, Zhan S, Kashanchi F, Fornace Jr AJ, Seth P, Helman LJ 1998 p53 regulates human insulin-like growth factor II gene expression through active P4 promoter in rhabdomyosarcoma cells. DNA Cell Biol 17:125–131[Medline]
  52. Ohlsson C, Kley N, Werner H, LeRoith D 1998 p53 regulates insulin-like growth factor-I (IGF-I) receptor expression and IGF-I-induced tyrosine phosphorylation in an osteosarcoma cell line: interaction between p53 and Sp1. Endocrinology 139:1101–1107[Abstract/Free Full Text]
  53. Prisco M, Hongo A, Rizzo MG, Sacchi A, Baserga R 1997 The insulin-like growth factor I receptor as a physiologically relevant target of p53 in apoptosis caused by interleukin-3 withdrawal. Mol Cell Biol 17:1084–1092[Abstract]
  54. Grimberg A 2003 Mechanisms by which IGF-I may promote cancer. Cancer Biol Ther 2:630–635[Medline]
  55. Oh Y, Muller HL, Lamson G, Rosenfeld RG 1993 Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition. J Biol Chem 268:14964–14971[Abstract/Free Full Text]
  56. Oh Y, Muller HL, Pham H, Rosenfeld RG 1993 Demonstration of receptors for insulin-like growth factor binding protein-3 on Hs578T human breast cancer cells. J Biol Chem 268:26045–26048[Abstract/Free Full Text]
  57. Liu B, Lee HY, Weinzimer SA, Powell DR, Clifford JL, Kurie JM, Cohen P 2000 Direct functional interactions between insulin-like growth factor-binding protein-3 and retinoid X receptor-{alpha} regulate transcriptional signaling and apoptosis. J Biol Chem 275:33607–33613[Abstract/Free Full Text]
  58. Grimberg A 2000 p53 and IGFBP-3: apoptosis and cancer protection. Mol Genet Metab 70:85–98[CrossRef][Medline]
  59. Sherr CJ 2004 Principles of tumor suppression. Cell 116:235–246[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
P. M. Yamada and K.-W. Lee
Perspectives in mammalian IGFBP-3 biology: local vs. systemic action
Am J Physiol Cell Physiol, May 1, 2009; 296(5): C954 - C976.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
R. Gallego, J. Codony-Servat, X. Garcia-Albeniz, E. Carcereny, R. Longaron, A. Oliveras, M. Tosca, J. M. Auge, P. Gascon, and J. Maurel
Serum IGF-I, IGFBP-3, and matrix metalloproteinase-7 levels and acquired chemo-resistance in advanced colorectal cancer
Endocr. Relat. Cancer, March 1, 2009; 16(1): 311 - 317.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
Y. Xi, G. Nakajima, T. Hamil, O. Fodstad, A. Riker, and J. Ju
Association of insulin-like growth factor binding protein-3 expression with melanoma progression
Mol. Cancer Ther., December 1, 2006; 5(12): 3078 - 3084.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M. Takaoka, C. E. Smith, M. K. Mashiba, T. Okawa, C. D. Andl, W. S. El-Deiry, and H. Nakagawa
EGF-mediated regulation of IGFBP-3 determines esophageal epithelial cellular response to IGF-I
Am J Physiol Gastrointest Liver Physiol, February 1, 2006; 290(2): G404 - G416.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
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 Grimberg, A.
Right arrow Articles by El-Deiry, W. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grimberg, A.
Right arrow Articles by El-Deiry, W. S.
Related Collections
Right arrow Metabolism

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