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Cell Growth & Differentiation Vol. 10, 49-59, January 1999
© 1999 American Association for Cancer Research

Retinoic Acid-mediated G1-S-Phase Arrest of Normal Human Mammary Epithelial Cells Is Independent of the Level of p53 Protein Expression1

Victoria L. Seewaldt2,, 3, Eric C. Dietze, Barton S. Johnson, Steven J. Collins and Molly B. Parker

Division of Oncology, Department of Medicine [V. L. S.], Department of Medicinal Chemistry [E. C. D.], Division of Hospital Dentistry, Department of Restorative Dentistry [B. S. J.], and Division of General Internal Medicine, University of Washington, Department of Medicine [M. B. P.], University of Washington, and Division of Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, Washington 98195 [S. J. C.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Retinoids mediate the normal growth of a variety of epithelial cells and may play an important role in the chemoprevention of breast cancer. Despite the widespread clinical use of retinoids, specific target genes that are regulated by retinoids are relatively poorly characterized. We reported previously that all-trans-retinoic acid (ATRA) mediates G1-S-phase arrest in normal human mammary epithelial cells (HMECs). The tumor suppressor gene p53 is thought to be a critical regulator of G1-S-phase arrest mediated by DNA-damaging agents such as chemotherapy and radiation. The role of p53 protein expression in G1-S-phase arrest mediated by the differentiating agent ATRA is unknown. Increased expression of p53 protein is observed in ATRA-treated HMECs at 72 h; however, initiation of G1-S-phase arrest starts at 24 h, suggesting that this observed induction of p53 is a secondary event. Using retroviral-mediated gene transfer, we expressed the E6 protein of the human papillomavirus strain 16 (HPV-16) in HMECs. The HPV-16 E6 protein binds to p53 and targets it for degradation. Western analysis confirmed that HPV-16 E6-transduced HMECs had markedly decreased levels of p53 protein expression. Suppression of cellular p53 levels in HMECs did not alter the sensitivity of HMECs to ATRA-mediated growth arrest. Our studies suggest that ATRA-mediated G1-S-phase arrest is independent of the level of p53 protein expression. We also tested the ability of estrogen and antiestrogens to induce growth arrest in HMECs lacking p53 expression and found no decrease in the sensitivity of these cells to these agents. Our results emphasize the chemotherapeutic potential of ATRA and antiestrogens, particularly for suppressing the growth of tumors lacking functional p53.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Retinoids are important for normal cellular growth and differentiation (1 , 2) and may play an important role in the chemoprevention of certain malignancies (3) . Retinoids can halt the progression of disease in premalignant lesions of the oral cavity, cervix, and skin and are effective in preventing the development of second primary tumors of the aerodigestive tract and lung (4, 5, 6, 7, 8, 9, 10, 11, 12, 13) . There is also evidence that retinoids are important for the prevention of breast cancer. The risk of breast cancer is increased for women with a lower dietary intake of vitamin A and ß-carotene, but not for women with dietary deficiencies of vitamins C or E (14) . Phase II trials are underway to test the ability of fenretinide, a synthetic retinoid, to prevent contralateral breast cancer (15 , 16) .

The actions of retinoids are thought to be mediated through specific nuclear RARs3 and retinoid X receptors belonging to the steroid/thyroid superfamily of transcription factors (17) . There are three RAR isoforms: RAR-{alpha}; -ß; and -{gamma}. Several lines of evidence suggest that the loss of expression of RAR-ß plays an important role in breast carcinogenesis. Whereas normal HMECs express RAR-ß mRNA, a majority of breast cancer cell lines fail to express this gene (18, 19, 20, 21) . Recently, it has been reported that there is a progressive decrease in RAR-ß mRNA levels during breast carcinogenesis (22 , 23) and a loss of heterozygosity on chromosome 3p24, which encodes the RAR-ß gene. It has also been observed that there is a loss of RAR-ß mRNA expression in breast cancer specimens as well as the morphologically normal-appearing adjacent tissue, but not in distal normal breast tissue (23 , 24) . These observations suggest that the loss of RAR function may be an important event in mammary carcinogenesis.

We reported previously that ATRA and RARs may act to regulate the G1-S-phase cell cycle transition in both normal and malignant mammary epithelial cells (20 , 21) . Regulation of G1-S-phase arrest in malignant cells by DNA-damaging agents such as radiation and chemotherapy has been well studied and is thought to involve the coordinated expression of specific cell cycle-regulatory proteins including p53. However, there is very little information on the regulation of the G1-S-phase cell cycle check point in normal cells by endogenous compounds such as retinoids. Understanding the molecular mechanism by which retinoids regulate proliferation will likely be important in the rational design of future retinoid-based chemoprevention trials (25) .

In this report, we investigate whether suppression of p53 protein expression in HMECs inhibits ATRA-mediated growth arrest. The E6 protein of the cancer-associated HPV-16 binds to p53 and targets it for degradation through the ubiquitin pathway (26 , 27) . Retroviral constructs containing HPV-16 E6 have been developed to investigate the role of p53 expression in regulating growth arrest mediated by DNA-damaging agents (28) . Using a similar approach, we expressed the HPV-16 E6 protein in HMECs and observed that unlike that with DNA-damaging agents, G1-S-phase arrest mediated by ATRA is independent of the level of p53 protein expression.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
p53 Expression in HMECs Undergoing ATRA-mediated G1-S-Phase Arrest.
Western blots were performed on parental HMEC strain AG11132 undergoing ATRA-mediated growth arrest to measure the temporal expression of p53 protein. AG11132 cells undergo G1-S-phase arrest starting 24 h after ATRA treatment (Fig. 1)Citation . As observed previously (21) , there was no induction of p53 protein at 24–48 h after ATRA treatment (Fig. 2)Citation . However, we did observe induction of p53 protein starting at 72 h after cells were treated with ATRA (Fig. 2)Citation .



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Fig. 1. Effect of RA on cell cycle phase distribution in HMECs. Cell cycle distribution of HMEC strain AG11132 (passage 11) treated with 1.0 µM ATRA. Cells were plated on day -1 in standard medium, re-fed on day 0, and treated with 1.0 µM ATRA on days 0, 1, 2, and 3. Cells were harvested on day 4 (see "Materials and Methods"). Data are presented relative to the %S- ({diamondsuit}) and %G1-(•)phase of the untreated cells and are representative of three separate experiments.

 


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Fig. 2. Induction of p53 protein expression is observed at 72 h in HMECs undergoing ATRA-mediated G1-S-phase arrest. AG11132-P parental cells (passage 11) were treated with 1.0 µM ATRA for 0, 1, 2, 3, and 4 days and analyzed for p53 protein expression as described in "Materials and Methods." Equal amounts of protein lysate were loaded in each lane. The protein gel was stained with Coomassie Blue, and a Mr 45,000 protein band was used as a loading control.

 
Retroviral-mediated Transduction of the HPV-E6 Antigen into Normal HMECs.
To investigate whether the suppression of p53 protein expression inhibits ATRA-mediated G1-S-phase arrest in HMECs, we used retroviral-mediated expression of the HPV-16 E6 protein to suppress normal intracellular p53 protein levels. The pLXSN16E6 retroviral vector containing the coding sequence for HPV-16 E6 viral protein has been described previously (28) . HMEC strain AG11132 is a heterogeneous population of normal HMECs derived from reduction mammoplasty (see "Materials and Methods"). These normal cells are growth factor dependent and undergo in vitro senescence after approximately 25 passages. Actively dividing passage 6 AG11132 cells were infected with retroviral vector LXSN16E6 or control vector LXSN, and the infected cells were selected as described in "Materials and Methods." Untransduced, parental AG11132 cells are referred to in this study as AG11132-P to distinguish them from transduced HMECs. Transduced cells expressing the HPV-16 E6 protein are designated AG11132-E6. Vector control clones are designated AG11132-LXSN. All experiments described hereafter were performed on mass cultures. AG11132-LXSN vector control cells, AG11132-P parental cells, and AG11132-E6 cells were serially passaged in culture. As observed previously, AG11132-LXSN vector control cells and AG11132-P parental cells exhibited a progressive increase in doubling time with continued serial passaging and underwent in vitro senescence at passage 24. In contrast, AG11132-E6 cells exhibited an extended life span in culture and did not undergo in vitro senescence until passage 40. This is not an unexpected result, because expression of the HPV-16 E6 protein under certain circumstances is sufficient to immortalize certain normal epithelial cells in vitro (29) .

Retroviral-mediated Expression of HPV-16 E6 Suppresses p53 Expression in HMECs.
Western blots were performed on AG11132-E6 cells and AG11132-LXSN vector controls (passage 11 and 35) to determine the relative levels of p53 protein. Expression of endogenous p53 protein was observed in AG11132-LXSN vector controls but was not detectable by Western analysis in AG11132-E6-transduced cells (Fig. 3)Citation .



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Fig. 3. Expression of p53, p21, cyclin D1/cdk4, and pRB protein in HMECs treated with ATRA. Passage 10 AG11132-LXSN vector controls, passage 10 AG11132-E6 transduced cells, and passage 27 AG11132-E6 transduced cells were treated for 48 h with (+) and without (-) 1.0 µM ATRA and analyzed for p53, p21, cyclin D1, cdk4, and pRB protein expression as described in "Materials and Methods." Equal amounts of protein lysate were loaded in each lane. The arrow denotes the location of the hyperphosphorylated pRB protein. The protein gel was stained with Coomassie Blue, and two Mr 65,000 (approximate) protein bands were used as a loading control.

 
HMECs Exhibiting Reduced Levels of p53 Protein Readily Undergo ATRA-mediated G1-S-Phase Arrest.
AG11132-E6-transduced cells and AG11132-LXSN vector control cells were grown in standard medium. ATRA inhibited the growth of both passage 10 AG11132-E6 cells (which exhibit markedly reduced cellular levels of p53 protein) and passage 10 AG11132-LXSN vector controls (Fig. 4A)Citation .



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Fig. 4. Suppression of p53 protein expression in HMECs does not inhibit ATRA-mediated growth inhibition. Growth curves of AG11132-LXSN vector controls and AG11132-E6 transduced cells at passage 10 (A), passage 18 (B), and passage 30 (C) treated with ({blacksquare}) and without ({diamondsuit}) 1.0 µM ATRA. AG11132-LXSN vector controls underwent in vitro senescence at passage 24; therefore, passage 30 growth curve data are available for AG11132-E6 only. Cells were plated on day -1 in standard medium in duplicate at 1 x 104 cells/well. Cells were re-fed on day 0 with standard medium containing 0 or 1.0 µM ATRA. Untreated controls received an equivalent volume of ethanol (0.1% final concentration). Cells were trypsinized at 24-h intervals and counted in triplicate. These data are representative of three separate experiments.

 
AG11132-P parental cells as well as AG11132-LXSN vector control cells underwent in vitro senescence associated with a progressive increase in doubling time as the cells were serially passaged in culture. Untreated AG11132-LXSN vector control cells exhibited a doubling time of approximately 24 h at passage 10 and 48 h at passage 18 and underwent terminal in vitro senescence at passage 24 (Fig. 4, A and B)Citation . In contrast, suppression of p53 protein expression by HPV-16 E6 protein in AG11132 cells extended the in vitro life span of these transduced cells to passage 40. Untreated AG11132-E6 cells exhibited a 24-h doubling time at passages 10 and 18 and a 48-h doubling time at passage 30 (Fig. 4, A, B, and C)Citation .

Despite the difference in the doubling times of untreated passage 18 AG11132-E6 and AG11132-LXSN vector control cells, both cell populations exhibited proportionally similar growth inhibition when treated with ATRA (Fig. 4BCitation ; Table 1Citation ). Passage 30 AG11132-E6 cells also retained sensitivity to ATRA (Fig. 4C)Citation . These data demonstrate that ATRA inhibits the proliferation of HMECs in culture, and that inhibition of p53 protein expression does not block the sensitivity of HMEC strain AG11132 to ATRA-mediated G1-S-phase arrest.


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Table 1 Suppression of p53 protein expression in HMECs does not effect ATRA-mediated growth inhibition

 
To further investigate whether intracellular p53 protein expression is critical for ATRA-mediated growth inhibition of HMECs, FACS analysis was performed on AG11132-E6 cells (passage 11–12) and AG11132-LXSN vector controls (passage 11–12) treated with 1.0 µM ATRA for 0, 1, 2, 3, and 4 days (Fig. 5Citation ; Table 2Citation ). Flow cytometric analysis of isolated nuclei stained with propidium iodide demonstrated that the suppression of p53 protein expression in AG11132-E6 cells did not alter their sensitivity to ATRA-mediated G1-S-phase arrest relative to AG11132-LXSN vector controls (Fig. 5Citation ; Table 2Citation ). These results also demonstrate that ATRA-mediated growth inhibition of HMEC strain AG11132 is not dependent on the level of p53 expression.



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Fig. 5. Suppression of p53 protein expression does not inhibit ATRA-mediated G1-S-phase arrest. Representative (days 0, 2, and 4) cell cycle histogram of HMEC AG11132-LXSN vector controls (passage 10; LXSN) and AG11132-E6 transduced cells (passage 10; E6) treated with 1.0 µM RA for 0, 1, 2, 3, and 4 days. Cells were plated on day -1 in standard medium, re-fed on day 0, and treated with 1.0 µM ATRA on days 0, 1, 2, and 3. Cells were harvested on day 4 (see "Materials and Methods"). The distribution of cells in the various phases of the cell cycle was determined by flow cytometry as described in "Materials and Methods." Data are representative of three separate experiments.

 

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Table 2 Suppression of p53 protein expression in HMECs does not inhibit ATRA-mediated G1-S-phase arrest

 
ATRA Does Not Mediate Apoptosis in AG11132-E6 Cells or in AG11132-LXSN Vector Controls.
We tested ATRA-treated AG11132-LXSN vector controls and AG11132-E6 cells for the presence of apoptosis by the terminal dexoynucleotidyl transferase method and by electron microscopy (data not shown). There was no evidence that ATRA mediated apoptosis in either AG11132-LXSN vector control cells or AG11132-E6 cells.

Expression of Cell Cycle Mediators in AG11132-E6 Cells Undergoing ATRA-mediated G1-S-Phase Arrest.
We performed Western analysis to further investigate whether the suppression of p53 protein expression in AG11132-E6 cells altered the expression of other cell cycle mediators in HMECs undergoing ATRA-mediated G1-S-phase arrest.

We investigated whether the suppression of p53 expression in AG11132-E6 cells (passage 11) resulted in a change in p21 protein expression in ATRA-treated and untreated cells. There is no induction of p21 protein expression when AG11132-LXSN vector controls and AG11132-E6 cells are treated with ATRA for 48 h (Fig. 3)Citation . However, there is a decrease in the absolute level of p21 expression in uninduced AG11132-E6 cells relative to controls (Fig. 3)Citation . These data suggest that the sensitivity of this strain of HMECs to ATRA-induced G1-S-phase arrest is not dependent on the absolute level of p21 protein expression, and that growth arrest mediated by ATRA does not involve the induction of p21.

We next investigated the expression of cyclin D1/cdk4 in AG11132-E6 cells (passage 11) and AG11132-LXSN vector controls undergoing ATRA-induced growth inhibition. Both AG11132-E6 cells and AG11132-LXSN vector controls exhibited a significant absolute decrease in cyclin D1 and cdk4 protein levels temporally associated with ATRA-mediated G1-S-phase arrest (Fig. 3)Citation . However, the relative level of cyclin D1 expression in AG11132-E6 cells was markedly reduced relative to that in AG11132-LXSN vector controls (Fig. 3)Citation . Whereas these results suggest that there is a temporal association between ATRA-mediated G1-S-phase arrest and decreased levels of cyclin D1/cdk4 protein, it does not appear that the absolute level of cyclin D1 expression is critical.

The tumor suppressor protein pRB seems to play a critical role in mediating cell cycle progression. We observe that ATRA-mediated G1-S-phase arrest of AG11132-LXSN vector controls and AG11132-E6 cells is temporally associated with a significant decrease in the level of hyperphosphorylated pRB concomitant with the induction of growth arrest (Fig. 3)Citation . These observations suggest that suppression of p53 does not alter the expression of pRB in HMECs, and that ATRA-induced G1-S-phase arrest in HMECs is associated with a decreased level of hyperphosphorylation, independent of the level of p53 expression.

AG11132-E6 cells have an extended life span in culture relative to vector controls. We also investigated the relative levels of these cell cycle mediators in AG11132-E6 cells at passage 35 as they neared senescence. There was a slight decrease in the levels of p21 and pRB protein expression in AG11132-E6 cells at passage 35 relative to passage 11; however, the overall pattern of expression of these cell cycle mediators as a function of ATRA treatment was unchanged as they approached senescence (Fig. 3)Citation .

Proteasome Inhibitors Block ATRA-mediated G1-S-Phase Arrest of HMECs and Associated Decrease in Cyclin D1 Protein Levels.
We next investigated whether blocking the function of the 26S proteasome antagonizes ATRA-mediated growth arrest of HMECs and the associated decline in cyclin D protein levels. AG11132-E6 and AG11132-LXSN vector controls were treated with calpain inhibitor I (1.0 µM) for 1.5 h before treatment with 1.0 µM ATRA. We observed that calpain inhibitor I blocked the ability of ATRA to induce growth arrest (Fig. 6A)Citation and also inhibited the expected decline in cyclin D1 levels (Fig. 7)Citation . This finding was confirmed and extended by treating our target cells with a second proteasome inhibitor, lactacystin (2.0 µM), which also prevented the ability of ATRA to induce growth arrest (Fig. 6B)Citation and a decline in cyclin D1 levels (Fig. 7)Citation . These data are consistent with the view that ATRA treatment decreases the level of cyclin D1 expression by promoting proteolysis via a ubiquitin-dependent pathway.



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Fig. 6. Proteasome inhibitors block ATRA-mediated growth inhibition. Growth curves of AG11132-LXSN vector controls and AG11132-E6 transduced cells treated with 1.0 µM calpain I inhibitor (A) and 2.0 µM lactacystin (B) before treatment with 1.0 µM ATRA. Cells were plated on day -1 in standard medium in duplicate at 1 x 104 cells/well. Cells were re-fed on day 0 with standard medium containing 1.0 µM calpain I inhibitor or 2.0 µM lactacystin for 1.5 h before treatment with either 0 or 1.0 µM ATRA. Untreated controls received an equivalent volume of ethanol (0.1% final concentration). Cells were trypsinized at 24-h intervals and counted in triplicate. These data are representative of three separate experiments.

 


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Fig. 7. Proteasome inhibitors block the expected decline of cyclin D1 protein in HMECs treated with ATRA. Passage 11 AG11132-LXSN vector controls and AG11132-E6 transduced cells were treated for 1.5 h with either 1.0 µM calpain I inhibitor or 2.0 µM lactacysin, treated for 48 h with (+) or without (-) 1.0 µM ATRA, and analyzed for cyclin D1 protein expression as described in "Materials and Methods." Equal amounts of protein lysate were loaded in each lane. The protein gel was stained with Coomassie Blue, and two Mr 65,000 protein bands were used as a loading control.

 
HMECs Exhibiting Reduced Levels of p53 Protein Undergo Growth Arrest when Treated with Antiestrogens.
Antiestrogens, similar to retinoids, have been observed to mediate growth arrest via a pRB-dependent pathway (30 , 31) . We tested whether inhibition of p53 expression would alter the sensitivity of HMEC strain AG11132 to the proliferative effects of estrogen and the growth-suppressive effects of antiestrogens TAM and OHT. AG11132-E6 transduced cells (with significantly reduced p53 expression) and AG11132-LXSN vector control cells were grown in standard medium or in Phenol Red-free media with a known concentration of E2 (5 nM). AG11132-E6 cells or AG11132-LXSN vector controls cells demonstrated a similar proliferative response to E2 (Fig. 8A)Citation . AG11132-E6 and AG11132-LXSN vector control cells were also cultured in standard media containing 1.0 µM TAM or 100 nM OHT. Inhibition of p53 expression did not reduce the sensitivity of HMEC strain AG11132 to the growth-inhibitory effects of the antiestrogens OHT (Fig. 8B)Citation and TAM (data not shown). FACs analysis confirmed that suppression of p53 protein expression did not inhibit OHT-mediated G1-S-phase arrest (Table 3)Citation .



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Fig. 8. Suppression of p53 protein expression does not alter the sensitivity of HMECs to the proliferative effects of E2 or growth inhibition by antiestrogens. Growth curves of AG11132-LXSN vector controls and AG11132-E6 transduced cells (passage 11; A) grown in either standard media or Phenol Red-free media with and without the addition of 5 nM E2 and (B) grown in standard media with or without the addition of 100 nM OHT. Synchronization of cells is described in "Materials and Methods." Cells were plated on day -1 in duplicate at 1 x 104 cells/well. Cells were treated on day 0. Untreated controls received an equivalent volume of ethanol (0.1% final concentration). Cells were trypsinized at 24-h intervals and counted in triplicate. These data are representative of three separate experiments.

 

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Table 3 Suppression of p53 protein expression in HMECs does not inhibit OHT-mediated G1-S-phase arrest

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Retinoids are important regulators of normal cellular proliferation and differentiation and have also been found to suppress tumor growth in vivo; however, little is known about their downstream molecular targets (1, 2, 3) . Understanding how retinoids work to regulate growth and differentiation is important for defining how retinoids may be used as chemopreventive agents and in combination with traditional chemotherapeutic agents.

Current molecular models of G1-S-phase arrest in mammalian cells have been primarily based on observations made in cells treated with DNA-damaging agents such as chemotherapeutic agents and ionizing radiation. In these model systems, G1-S-phase arrest was associated with the coordinated induction of p53 and p21 that subsequently regulated the activity of cyclin/cdk complexes and resulted in pRB hypophosphorylation (32, 33, 34, 35, 36, 37, 38, 39) . Suppression of p53 protein expression in human keratinocytes by HPV-16 E6 correlated with an inability of these cells to undergo growth arrest mediated by DNA-damaging agents (37) . Whereas p53 may play a pivotal role in regulating the growth-suppressive actions of DNA-damaging agents in malignant cells, it is unclear what role it may play in growth regulation mediated by endogenous hormones, vitamins, and cytokines in normal cells. It has recently, been reported that certain physiological signals that inhibit cell growth such as transforming growth factor ß or prostaglandin A2 may regulate pRB phosphorylation independent of p53 expression (40 , 41) . We hypothesized that ATRA may similarly regulate cell growth via a p53-independent pathway.

As observed previously, there is no temporal correlation between the initiation of ATRA-mediated G1-S-phase arrest in normal cells and the induction of p53 protein (Figs. 1Citation and 2Citation ; Ref. 21 ). G1-S-phase arrest mediated by ATRA starts at 24 h (Fig. 1)Citation , and there is no induction of p53 protein until 72 h after treatment (Fig. 2)Citation , when a majority of cells have undergone growth arrest (Fig. 1)Citation . Based on these observations, we hypothesized that p53 protein induction did not play a primary role in regulating ATRA-mediated G1-S-phase arrest, but we could not exclude the possibility that p53 expression played a permissive role.

To test whether p53 expression is critical for ATRA-mediated G1-S-phase arrest in normal cells, we suppressed p53 protein expression in HMECs using retroviral-mediated expression of the HPV-16 E6 protein. Expression of the HPV-16 E6 protein in HMEC strain AG11132 markedly reduced the level of intracellular p53 (Fig. 3)Citation . No correlation was found between the level of p53 expression and the ability of ATRA to induce growth inhibition (Fig. 4Citation ; Table 1Citation ) and G1-S-phase arrest (Table 2Citation ; Fig. 5Citation ).

Similar to retinoids, estrogens and antiestrogens have been observed to regulate proliferation via a pRB-dependent pathway (30 , 31 , 42) . We tested the sensitivity of HMEC strain AG11132 to the proliferative effects of E2 and the growth-inhibitory effects of the antiestrogens TAM and OHT and observed that there was no decreased sensitivity to the effects of estrogens or antiestrogens in cells with decreased p53 levels (Fig. 8)Citation . These observations suggest that unlike DNA-damaging agents, growth regulation by endogenous compounds such as ATRA and estrogens does not depend on the level of p53 protein expression.

Biochemical analysis of untreated AG11132-E6 cells showed a significant decrease in the absolute levels of p21 and cyclin D1 relative to AG11132-LXSN vector controls (Fig. 1)Citation . The observed decrease in p21 protein levels in AG11132-E6 cells is an expected finding, because the p53 protein positively regulates the transcription of p21 (29) . The mechanism by which a decrease in intracellular p53 protein levels results in a decrease in the level of cyclin D1 protein is less clear. AG11132-LXSN and AG11132-E6 cells demonstrate equal sensitivity to the growth-inhibitory effects of ATRA, suggesting that G1-S-phase arrest mediated by ATRA is independent of the absolute levels of p21 and cyclin D1 expression. However, cyclin D1 levels decline in both ATRA-treated AG11132-E6 and AG11132-LXSN cells (relative to untreated cells), suggesting that a decline in cyclin D1 expression may play a role in ATRA-mediated growth inhibition.

Overexpression and amplification of cyclin D1 have been observed in primary breast cancers, even at early stages of the disease (43 , 44) , and it is hypothesized that lack of coordinated expression of cyclin D1 might promote breast carcinogenesis. Dysregulated expression of cyclin D1 in the human breast cancer cell line T-47D induces progression from G1 to S phase (45) , and overexpression of cyclin D1 in breast cancer cells decreases the rate at which cells exit the cell cycle, allowing growth factor-independent cell cycle progression and pRB hyperphosphorylation (46) . These observations predict that dysregulated expression of exogenous cyclin D1 would result in retinoid resistance. However, ectopic expression of cyclin D1 in BEAS-2G immortalized bronchial epithelial cells did not alter the sensitivity of transductants to retinoid-mediated growth arrest (47) . Regulation of cyclin D1 protein levels by retinoids seems to involve posttranslational proteolysis. We observe that proteosome inhibitors calpain inhibitor I and lactacystin both inhibit the ability of ATRA to mediate G1-S-phase arrest and prevent the expected associated decline in cyclin D1 protein levels. These observations are similar to the reported response of BEAS-2G cells to calpain I inhibitor and lactacystin (47) .

p53-mediated G1-S-phase arrest requires the coordinated expression of a complex set of cell cycle-regulatory genes. At the present time, the precise molecular pathway by which ATRA mediates G1-S-phase arrest in is unknown. We observe that ATRA-mediated growth arrest in HMECs does not depend on the level of p53 protein expression. It is unclear, however, whether growth arrest mediated by ATRA uses the same downstream effectors as p53 or unique downstream effectors, or whether there is an overlap between the two pathways. The data presented here suggest that there may be some overlap between the two pathways, because there is a decrease in the levels of cyclin D1, cdk4, and hyperphosphorylated pRB temporally associated with ATRA-mediated G1-S-phase arrest, similar to p53-dependent growth arrest. There also seems to be cross-talk between the downstream effector targets of p53 and ATRA because (a) inhibition of p53 protein expression results in a decrease in the absolute levels of cyclin D1; and (b) ATRA regulates the expression of cyclin D1, cdk4, and hyperphosphorylated pRB. Additional experiments will be necessary to define the potential interaction between downstream mediators of p53- and ATRA-mediated G1-S-phase arrest.

The sensitivity of AG11132-E6 cells to ATRA-mediated growth inhibition despite the suppression of p53 expression suggests that retinoids might have chemotherapeutic potential for suppressing the growth of tumors lacking p53 function. These results suggest novel potential clinical applications for retinoids in the treatment of tumors that contain p53 mutations. It is interesting that a naturally occurring dietary compound is able to induce growth regulation independent of p53 expression, highlighting the importance of investigating physiological signal agents as potential chemoprevention and therapeutic agents.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Materials.
ATRA (Sigma, St. Louis, MO), E2 (Sigma), TAM (Sigma), and OHT (Sigma) stock solutions were prepared in 100% ethanol and stored in opaque tubes at -70°C. Control cultures received equivalent volumes of the ethanol solvent. RA stocks were used under reduced light. The proteasome inhibitors calpain inhibitor I (Calbiochem, Nova Biochem, La Jolla, CA) and lactacystin (Calbiochem) were used.

Cell Culture and Media.
Normal HMEC strain AG11132 (M. Stampfer 172R/AA7) was purchased from the National Institute of Aging, Cell Culture Repository (Coriell Institute; Ref. 48 ). HMEC strain AG11132 was derived from normal mammary tissue obtained at reduction mammoplasty, has a limited life span in culture, and fails to divide after approximately 25 passages. AG11132 was at passage 8 at the time of receipt. Cells were grown in Mammary Epithelial Cell Basal Medium (Clonetics, San Diego, CA) supplemented with bovine pituitary extract (Clonetics CC4009; 4 µl/ml), insulin (Lake Placid, NY; 5 µg/ml), epidermal growth factor (Upstate Biotechnology, Inc.; 10 ng/ml), hydrocortisone (Sigma; 0.5 µg/ml), isoproterenol (Sigma; 10-5 M), and HEPES buffer (Sigma; 10 mM). G418 (Life Technologies, Inc., Grand Island, NY)-containing media was prepared by the addition of 300 µg/ml G418 to the standard media described above. Cells were cultured at 37°C in a humidified incubator with 5% CO2/95% air. We did not process our growth media to remove endogenous retinoids. Mycoplasma testing was performed as reported previously by Russell et al. (49) .

Retroviral Transduction.
The LXSN16E6 retroviral vector containing the HPV-16 E6 coding sequence (provided by D. Galloway, Fred Hutchinson Cancer Research Center, Seattle, WA) has been described previously (29) . AG11132 normal HMECs (passage 9) were plated in four T-75 tissue culture flasks (Corning) in standard medium and grown to 50% confluence. Transducing virions from either the PA317-LXSN16E6 or control PA317-LXSN (without insert) retroviral producer line were added at a multiplicity of infection at 1:1 in the presence of 4 µg/ml Polybrene (Sigma) to log-phase cells grown in T-75 flasks. The two remaining T-75 flasks were not infected with virus. After 48 h, the two flasks containing transduced cells and one flask with untransduced cells were selected with standard medium containing G418 (300 µg/ml). Cells were continued in G418-containing medium for 2 weeks until 100% of control untransduced cells were dead. The fourth flask of unselected, untransduced parental control cells was passaged in parallel with the selected, transduced experimental and vector control cells.

ATRA Cell Growth Curves.
AG11132-LXSN vector controls (passage 10 and 18) and AG11132-E6 transduced cells (passages 10, 18, and 30) were plated in duplicate at 1 x 104 cells/well in 12-well tissue culture plates (Corning) on day -1 and allowed to adhere. On day 0, the medium was replaced with standard medium with or without 1.0 µM ATRA. Untreated controls received an equivalent volume of ethanol solvent (0.1% final concentration). Cells were trypsinized at 24-h intervals and counted in triplicate.

Estrogen Proliferation Assays.
AG11132-LXSN vector controls (passage 10 or 11) and AG11132-E6 transduced cells (passages 10 or 11) were plated on day -4. On day -3, media were changed to Phenol Red-free Mammary Epithelial Cell Basal Medium (Clonetics) with standard media additives for 48 h. Cells were plated on day -1 in duplicate at 1 x 104 cells/well in 12-well tissue culture plates (Corning) and allowed to adhere. On day 0, the media was changed to either Phenol Red-free Mammary Epithelial Cell Basal Media or standard media with or without 5 nM E2. Cells were trypsinized at 24-h intervals and counted in triplicate.

Antiestrogen Cell Growth Curves.
AG11132-LXSN vector controls (passage 10) and AG11132-E6 transduced cells (passage 10) were plated in duplicate at 1 x 104 cells/well in 12-well tissue culture plates (Corning) on day -1 and allowed to adhere. On day 0, the medium was replaced with standard medium with or without 1.0 µM TAM or 100 nM OHT. Untreated controls received an equivalent volume of ethanol solvent (0.1% final concentration). Cells were trypsinized at 24-h intervals and counted in triplicate.

Proteasome Inhibitor Growth Curves.
AG11132-LXSN vector controls (passage 10) and AG11132-E6 transduced cells (passage 10) were plated in duplicate at 1 x 104 cells/well in 12-well tissue culture plates (Corning) on day -1 and allowed to adhere. On day 0, the medium was replaced with standard medium with or without 1.0 µM calpain inhibitor I or 2.0 µM lactacystin. After 1.5 h, cells were treated with or without 1.0 µM ATRA. Cells were trypsinized at 24-h intervals and counted in triplicate.

DNA Staining of Cell Nuclei with Propidium Iodide and FACS Analysis.
A total of 5 x 105 AG11132-P (passage 10), AG11132-LXSN (passage 10–12), or AG11132-E6 cells (passage 10–12) were plated in T-75 flasks (Corning) on day -1 and allowed to adhere. On day 0, the medium was removed and replaced with 20 ml of fresh medium. ATRA was added to the culture medium for a final concentration of 1.0 µM on days 0, 1, 2, and 3 for the preparation of day 4, 3, 2, and 1 time points, respectively. Cells were harvested on day 4 and did not exceed {approx}70% confluence. Cells were trypsinized, and nuclei were isolated and stained with propidium iodide as reported previously (20) . Nuclei were then analyzed by FACScan (20) . Ten thousand events were collected in list mode fashion, stored, and analyzed on Muticycle AV software (Phoenix Flow Systems, San Diego, CA). Cells were treated similarly with either 100 nM OHT or 1.0 µM TAM, and nuclei were isolated and analyzed by FACScan.

Western Blotting.
Preparation of cellular lysates and immunoblotting were as described previously (21) . Equal amounts of protein lysates (approximately 100 µg of total protein) were loaded on 10% polyacrylamide gels, and the gels were run and then electroblotted (Hoeffer) at 80 mA for 45 min onto Hybond enhanced chemiluminescence membrane (Amersham). The membrane was blocked with 20% BSA (Sigma) in PBS overnight at RT and then incubated with either a 1:100 dilution of mouse antihuman p53 (Oncogene Science Ab-2), a 1:100 dilution of mouse antihuman pRB antibody (PharMingen), a 1:100 dilution of mouse antihuman p21/WAF1 antibody (Santa Cruz Biotechnology), a 1:200 dilution of polyclonal rabbit antiserum against human cyclin D1 (Santa Cruz Biotechnology), or a 1:100 dilution of polyclonal mouse antiserum to human cdk4 (Santa Cruz Biotechnology) for 1 h at RT with agitation. The membrane was washed three to five times at RT with 250 ml of PBS containing 0.1% Tween and then incubated with either a horseradish peroxidase-conjugated goat antimouse IgG (Jackson ImmunoResearch) at a 1:35,000 dilution or a 1:2,000 dilution of horseradish peroxidase-conjugated protein A (Sigma) for 1 h at RT. The blot was washed again, and complexes were detected by using enhanced chemiluminescence Western blotting detection reagents (Amersham) as described by the manufacturer.


    Acknowledgments
 
We acknowledge the generous gift of LXSN16E6 retroviral supernatant from Denise Galloway.


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

1 Supported by a Pilot and Feasibility Award from the Clinical Research Unit, University of Washington (Seattle, WA), NIH National Institutes of Diabetes, Digestive and Kidney Disease, and 2P30 DK 35816-11 [to V. L. S.]. V. L. S. was supported by NIH K08 Clinical Investigator Award CA 68210-01, a New Investigator Award from the Clinical Nutrition Research Unit NIH National Institutes of Diabetes, Digestive and Kidney Disease, 2P30DK 35816-11, and a Susan G. Komen Breast Cancer Fellowship. Back

2 To whom requests for reprints should be addressed, at Arthur James Cancer Center, Ohio State University, A437A Starling Loving Hall, Columbus, OH 43210. Phone (614) 293-4766; Fax: (614) 293-7525; E-mail: seewaldt-1{at}medctr.osu.edu Back

3 The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; RA, retinoic acid; ATRA, all-trans-retinoic acid; HMEC, human mammary epithelial cell; HPV-16, human papillomavirus type 16; RARE, RA response element; OHT, 4-OH-tamoxifen, TAM, tamoxifen, E2, 17-ß-estradiol, ECL, enhanced chemiluminescence; FACS, fluorescence-activated cell sorting; RT, room temperature; cdk, cyclin-dependent kinase. Back

Received for publication 3/24/98. Revision received 11/10/98. Accepted for publication 11/12/98.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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