Carcinogenesis, Vol. 20, No. 7, 1161-1168,
July 1999
© 1999 Oxford University Press
Accelerated Paper |
Taxol, vincristine or nocodazole induces lethality in G1-checkpoint-defective human astrocytoma U373MG cells by triggering hyperploid progression
Department of Head and Neck Surgery, University of Texas M.D. Anderson Cancer Center, Box 69, 1515 Holcombe Boulevard, Houston, TX 77030,
1 Abbott Laboratories, PPD, D-460, AP10-1, 100 Abbott Park Road, Abbott Park, IL 60064 and
2 University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA
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Abstract |
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In this report, we describe a novel lytic mechanism exploited by antimicrotubule drugs (AMDs) such as Taxol which are frequently used to treat multiple human cancers including breast and ovarian cancers. In cells lacking the G1-arresting capacity due to the defect in retinoblastoma or p53 gene function, AMDs trigger hyperploid progression and death. The hyperploid progression occurs via continued cell-cycle progression without cell division. Blocking hyperploid progression through hydroxyurea or ectopically expressed p27Kip1, a G1-specific Cdk inhibitor, abrogates AMD cytotoxicity. Thus, AMDs induce lethality in G1-checkpoint-defective cells by triggering hyperploid progression. The phenomenon is reminiscent of that observed previously with bub-1 yeast mutant. The potential significance of this finding lies in that hyperploid progression-mediated death may be exploited to develop a therapy with tumor-specificity at the genetic level. As a large fraction of human cancers are mutated in p53 gene, it may have a wide therapeutic applicability.
Abbreviations: AMD, antimicrotubule drug; MEF, mouse embryo fibroblast; MT, microtubule; RB, retinoblastoma.
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Introduction |
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Antimicrotubule drugs (AMDs) represent a critical arsenal against human cancers. AMDs include nocodazole, which inhibits the addition of tubulin molecules to microtubules (MTs) leading to MT depolymerization; vincristine, which induces the formation of paracrystalline aggregates of tubulin leading to MT depolymerization; and Taxol, which binds tightly to polymers and inhibits MT depolymerization (1–3). Among them, Taxol and vincristine have been used effectively against advanced stage breast and ovarian cancers (4–8). Despite its effectiveness, the major problem facing AMD chemotherapy stems from the multiple adverse reactions it triggers in patients, which include bone marrow suppression, neutropenia, leuokopenia and anemia (4–8). To circumvent these problems, it is vital to understand the mechanism through which AMDs induce lethality in cancer cells in order to exploit the mechanism to develop a tumor-specific therapy.
Several reports have been made recently that shed light on the events following MT disruption by AMDs. In yeast, disruption of MTs by AMDs leads to a defect in the mitotic spindle, which triggers an M-phase arrest due to the spindle checkpoint that monitors various aspects of the machinery involved in chromosome segregation (9,10). In mammalian cells, a similar scenario unfolds, except that the M-phase arrest is transient and the AMD-treated cells become G1-arrested after re-entering the cell cycle (11–17). In mouse embryo fibroblast (MEF) mutants lacking retinoblastoma (RB) or p53 tumor suppressor gene function, however, the G1-arrest fails to occur and the AMD-treated cells become hyperploid (12,13). A similar result was also obtained after inactivating RB, p53 or p21Waf1 gene function in several human fibroblasts and cell lines (15–17).
In this report, we investigated whether the triggering of hyperploid progression leads to death. Previously, we reported that U373MG human astrocytoma cells undergo hyperploid progression upon treatment with the AMD nocodazole (14). The p53 gene of U373MG cells contains an Arg to His mutation in codon 273 (18), which represents the second most frequently occurring p53 mutation in human cancers (19). Our results indicate that inhibiting hyperploid progression abrogates the cytotoxicity of multiple AMDs including Taxol and vincristine. The phenomenon is reminiscent of that observed previously with bub-1 budding yeast mutant (9), suggesting that the lytic pathway has been conserved. The potential significance of this finding lies in that the hyperploid-progression-mediated death may be exploited to develop a therapy with tumor specificity at the genetic level. As a large fraction of human cancers are mutated in the p53 gene, it may have a wide therapeutic applicability.
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Materials and methods |
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Cell culture
Human astrocytoma cells U373MG, SW1088 and CCF-STTG1 and human glioblastoma cells U-87MG were obtained from the American Type Culture Collection (Bethesda, MD). Normal human fibroblasts AG10803, AG09309 and AG06103A were obtained from the Coriell Cell Culture Facility of the National Institute of Aging (Bethesda, MD). U373MG and CCF-STTG1 cells were maintained in RPMI (with 10% FBS), U-87MG cells in MEM (with 10% FBS) and SW1088 cells in Leibovitz's L-15 medium (with 10% FBS) at 37°C in 5% CO2 environment. Media were obtained from Gibco BRL (Gaithersberg, MD). Cells were treated with Taxol (paclitaxel: 0.014 µM), vincristine (0.040 µM), nocodazole (0.415 µM) or hydroxyurea (3 mM) purchased from Sigma (St Louis, MO).
Flow cytometry
Flow cytometry was performed as described (14). Briefly, cells (5x105) were plated in 60 mm plates overnight and treated with drugs as indicated. Cells were harvested using trypsin and fixed in 70% ethanol for 16 h at 4°C. Propidium iodide was added (100 Kunitz units/ml) with RNase A (50 µg/ml) and DNA content per cell was measured using a Beckton Dickinson Fluorescence Activated Cell Sorter (FACS, Franklin Lakes, NJ). A Lysis II program was used to quantify cell cycle distribution. Ploidy was measured by modal chromosome content per cell. Dead cells were excluded from FACS analysis by gating only viable cells. To isolate G0/G1 phase cells, asynchronously grown cultures were trypsinized, rinsed with PBS, incubated with Hoechst dye 33342 (5 µg/ml) in PBS for 30 min at 25°C and flow-sorted using a Beckton Dickinson FACS PLUS flow cytometer. After sorting, cells were rinsed with PBS prior to reincubation in culture medium.
RB protein detection
Western blot analysis was performed as described (20). Cells were suspended in sample buffer (2% SDS, 20% glycerol, 0.12 M Tris pH 6.8), sonicated and boiled for 2 min. After centrifuging at 12 000 r.p.m. to remove debris, the lysate was separated by 6% SDS–PAGE and transferred to Immobilon-P nitrocellulose paper. Western blot analysis was performed using monoclonal anti-human RB antibody 14001A (Pharmingen, La Jolla, CA) as the primary antibody, followed by goat polyclonal HRP-conjugated anti-mouse antibody (Boehringer Mannheim, Indianapolis, IN) as the secondary antibody. The resulting blot was visualized using an enhanced chemiluminescence detection kit from Amersham Life Science (Arlington Heights, IL).
Cdk activity assay
Cdk2 kinase activity assay was performed as described (14). Cells were scraped from plates, resuspended in lysis buffer (50 mM Tris pH 7.4, 250 mM NaCl, 5 mM EDTA, 0.1 mM NaF, 0.1 mM Na2VO3, 1 mM PMSF, 0.5% NP-40), sonicated, and spun to remove debris. Cdk2 was immunoprecipitated by adding non-crossreactive (against other Cdks) anti-human Cdk2 goat polyclonal antibody 163-G (Santa Cruz Biotech, Santa Cruz, CA), recognizing amino acids 283–298, for 1 h and then incubating with protein A–Sepharose for 1 h at 4°C. The resulting immunoprecipitate was washed five times with lysis buffer. Kinase reaction was preformed at 30°C for 30 min in a buffer composed of 50 mM Tris (pH 7.4), 10 mM MgCl2, 5 mM MnCl2 and 1 mM DTT containing 0.5 µg purified histone H1 (Calbiochem, La Jolla, CA) and 0.1 µCi [
-32P]ATP. Cdc2 activity was assayed similarly using non-crossreactive anti-human Cdc2 rabbit polyclonal antibody 954 (Santa Cruz Biotech) recognizing amino acids 278–297. The resulting reaction mix was separated by 10% SDS–PAGE, transferred to a 3M Whatman paper, dried and autoradiographed.
[3H]thymidine incorporation assay
U373MG cells (1x104 cells per well) grown in 96-well plates were synchronized at the G1/S boundary by incubating with hydroxyurea (3 mM) for 48 h. Synchronization was confirmed by the absence of BrdU incorporation. After release from synchrony, cells were immediately incubated in medium containing nocodazole and [3H]thymidine (0.2 µCi) for 8 h intervals, which represents one-sixth of the cell cycle. After labeling, cells were fixed in 75% methanol–25% acetic acid for 10 min, washed with 10% TCA and dissolved in 0.2 N NaOH at 37°C for 12 h. Incorporated radioactivity was determined using a Packard 1990CA liquid scintillation counter (Downers Grove, IL). Experiments were performed in triplicate.
BrdU and 4',6'-diamidino-2-phenylindone (DAPI) immunofluorescence assays
BrdU incorporation assays were performed by incubating cells in eight-chamber slides and labeling with BrdU (0.1 mM) during the final 24 h of incubation prior to fixing in 70% ethanol at 4°C for 12 h. Fixed cells were denatured in 2 N HCl plus 0.5% Triton X-100 for 30 min, rinsed with PBS and incubated in a staining solution [5 µg/ml of FITC-conjugated monoclonal anti-BrdU antibody 1202–693 (Boehringer Mannheim) in PBS] for 30 min. After destaining in PBS for 10 min, cells were viewed with a Nikon Lobophot immunofluorescence microscope. To stain with DAPI, cells were fixed in methanol containing DAPI (2 µg/ml) for 10 min and destained for 12 h in methanol prior to viewing.
Cytogenetics
romosomes were prepared as described (21). Briefly, cells were dissociated using trypsin–EDTA, incubated in a solution containing 37 mM KCl and 400 mM sodium citrate for 30 min at 37°C and fixed at –20°C for 12 h. Chromosome preparations were stained with Giemsa.
Adenovirus construction and infection
The procedure used to construct Ad-p27 (expressing human p27Kip1) and Ad-ß-gal (expressing prokaryotic ß-galactosidase) recombinant adenoviruses was described previously (14). Infection with the recombinant adenoviruses was performed as described (14). U373MG cells were infected at a multiplicity of infection (m.o.i.) of 10 for 3 h in RPMI media containing 2% FBS.
Growth curves
Cells were plated at a density of 5x104 cells per 60 mm plate. Viable cells were counted using a hemocytometer. Viability was determined by exclusion of trypan blue dye. Experiments were performed in triplicate.
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Results |
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AMDs trigger hyperploid progression
Previously, we showed that mitotic spindle disruption by the AMD nocodazole, leads to hyperploid cell accumulation in human astrocytoma U373MG cells (14). Flow cytometry of nocodazole-treated cells shows that there is an increase of cells in G2/M phase, followed by a decline in this population with the appearance of hyperploid cells (Figure 1A
). Taxol and vincristine, two clinically used AMDs also induced hyperploid progression (data not shown). Nocodazole also induced hyperploidy in WERI-1 RB, CCF-STTG1 astrocytoma, SW1088 astrocytoma, Saos-2 osteosarcoma and U87MG glioblastoma cells (data not shown). In contrast, hyperploid progression did not occur in nocodazole-treated normal human fibroblasts including AG10803 (Figure 1A), AG09309 and AG06103A. Normal human brain cells were not available for this experiment. Continued treatment of U373MG cells with nocodazole led to the death of the hyperploid cells as indicated by the decrease in hyperploidy peak (Figure 1C).
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To confirm that the hyperploid cells observed after treating with nocodazole are derived from `2N' cells (U373MG contains a modal chromosome number of 60–69 chromosomes per cell but will be refered to as 2N for the rest of paper) rather than through the expansion of pre-existing hyperploid cells, the experiment was repeated using flow-sorted G0/G1-phase U373MG cells. G0/G1-phase cells were sorted to avoid contamination by pre-existing hyperploid cells. Asynchronously grown U373MG cells were labeled with Hoechst dye 33342, which binds non-covalently to AT-rich dsDNA, and sorted using a flow cytometer. Nocodazole treatment of the sorted G0/G1-phase cells led to hyperploid cell accumulation, confirming that they arose from 2N cells (Figure 1B
). Hyperploid cell accumulation did not occur in cells that were similarly sorted but incubated without nocodazole, indicating that this is not an artifact of cell sorting (Figure 1B).
Cytogenetic analysis of hyperploid cells
Nocodazole-treated U373MG cells were cytogenetically analyzed. The treatment led to the disappearance of cells containing an original average chromosome number of 60–69 and the appearance of cells containing 110–120 (for the majority) or 223–230 chromosomes (Figure 2). Karyotypes revealed that the majority of chromosomes underwent a uniform increase in copy number following nocodazole treatment. This is seen most clearly with the recognizable isochromosome 8q (denoted with arrowheads in Figure 2). Thus, nocodazole treatment induces polyploidy in U373MG cells. Endoreduplication, which occurs via bypassing of M phase despite the repeated S-phase entries resulting in polyvalent chromosomes (22), as the underlying mechanism was excluded as the chromosomes of nocodazole-treated U373MG cells were bivalent.
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The mechanism of hyperploid progression
To determine the mechanism through which hyperploid progression occurs in U373MG cells, nocodazole-treated U373MG cells were analyzed both cellularly and biochemically. Microscopic observations indicate that nocodazole-treated U373MG cells are blocked from undergoing cell division. After treatment with nocodazole for 24 h, the majority of U373MG cells became mitotically arrested with rounded and refractile morphology (Figure 3A
), which failed to undergo cell division even after 96 h.
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To gain insight into the cell-cycle status of hyperploid cells, we initially examined the phosphorylation pattern of endogenous RB protein. RB is a nuclear protein that is hypophosphorylated in G1, increasingly phosphorylated in S and G2 and hyperphosphorylated in M phase (20). Western blot analysis of nocodazole-treated U373MG cells showed both hyperphosphorylated and unphosphorylated forms of RB protein (Figure 3B
), indicative of cells progressing through the cell cycle. In contrast, normal human fibroblasts, which show principally hypophosphorylated RB due to their slow-dividing nature, exhibited the accumulation of hyperphosphorylated RB after treatment with nocodazole, indicative of their M arrest.
If the nocodazole-treated U373MG cells were to re-enter the cell cycle, then their exit from M phase should be detectable. To document the exit from M phase, their Cdc2 activity was monitored. Activation of Cdc2, which occurs during late G2 and lasts till the end of M phase, is required for M-phase entry in mammalian cells (23). Synchronized U373MG cells were released into nocodazole-containing medium and assayed for Cdc2 activity periodically. After ~24 h, Cdc2 activity reached its highest level, which persisted for ~32 h, indicative of their M-phase arrest (Figure 4). The heightened activity then declined, indicative of their exit from M phase after a transient arrest.
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If hyperploid progression occurs through continued cell cycling without cell division, then one should be able to block it by expressing G1-specific Cdk inhibitors. p27Kip1 is an inhibitor of cyclin-dependent kinases Cdk4 and Cdk6 controlling passage through early G1, as well as Cdk2 controlling progression through late G1 phase (23). Figure 5A
shows that ectopic expression of p27Kip1 (mediated through infection with recombinant adenovirus Ad-p27) in nocodazole-treated U373MG cells resulted in the absence of a hyperploid peak along with an increase in the G2/M peak. The expression of p27Kip1 following Ad-p27 infection was confirmed by western blot analysis (14). That the increase in the G2/M peak is due to cells that have traversed through M phase but became arrested at G1 phase was confirmed by the appearance of unphosphorylated RB specific to G1 phase (Figure 5B), suppression of Cdk2 activity present in nocodazole-treated cells (Figure 5C) and the lack of BrdU incorporation (Figure 5D). No such effect was observed with Ad-ß-gal infected, nocodazole-treated cells (Figure 5A–D). Thus, ectopically expressed p27Kip1 could block hyperploid progression by inducing G1 arrest in `4N' cells that have re-entered the cell cycle without the cell division.
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To detect re-entry into S phase during hyperploid progression, a [3H]thymidine incorporation assay was performed. Synchronized U373MG cells were released into nocodazole-containing medium and labeled with [3H]thymidine regularly. Soon after the release, an intense [3H]thymidine incorporation occurred indicative of first S phase (Figure 6A
). After a period slightly longer than the cycling time of 44 h, another incorporation peak occurred, indicating the onset of second S phase. Ongoing DNA synthesis in nocodazole-treated U373MG cells was confirmed through BrdU incorporation (Figure 6B). The reduced intensity of the second peak is probably due to nododazole cytotoxicity.
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Next, the effect of blocking DNA synthesis on hyperploid progression was examined. Simultaneous treatment with nocodazole and hydroxyurea resulted in G0/G1 and G2/M peaks with little progression to hyperploidy (Figure 6C
, panel 2). The occurrence of the G2/M peak, together with the absence of a hyperploid peak, suggests that 4N cells that have exited M phase are blocked by hydroxyurea from re-entering the S phase neccessary for hyperploid progression. This was confirmed by the corresponding increase in hyperploid cells observed after delaying hydroxyurea co-treatment to 24 or 48 h (Figure 6C, panels 3 and 4). The results thus indicate that DNA synthesis is required for hyperploid progression. Taken together, our results indicate that the nocodazole-treated U373MG cells exit from M phase despite the lack of cell division and re-enter G1 and S phases during hyperploid progression.
Taxol, vincristine or nocodazole induces lethality in U373MG cells by triggering hyperploid progression
Next, the potential relevance of inducing hyperploid progression on AMD cytotoxicity was investigated. To this end, U373MG cells were treated with nocodazole, and cells were tested for trypan blue dye exclusion to assay viability and flow cytometry to determine DNA content over time. Figure 7A shows that hyperploid progression correlated with cytotoxicity, indicating that the two processes are linked.
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If triggering hyperploid progression leads to death, then inhibition of cell cycling required for hyperploid progression should block AMD cytotoxicity. To test this, asynchronously grown U373MG cells were co-treated with nocodazole and hydroxyurea and viability was determined. Simultaneous co-treatment blocked nocodazole cytotoxicity almost completely (Figure 7B
). Flow cytometry of similarly treated cells showed the presence of cells in the G2/M peak (Figure 6C, panel 2). In the preceding section, we showed that the G2/M peak in nocodazole and hydroxyurea co-treated cells represents 4N cells that have exited M phase but are subsequently arrested at the G1/S boundary by hydroxyurea. This indicated that neither the lack of cell division nor the inappropriate re-entry into G1 phase occurring without cell division is sufficient to induce death by nocodazole. To determine the effects of allowing hyperploid progression in nocodazole-treated cells, hydroxyurea treatment was delayed by 24 or 48 h. The cytotoxic effects of nocodazole resumed, as evidenced by the decline in viability until hydroxyurea was added (Figure 7B). The occurrence of hyperploidy in these cells was confirmed by flow cytometry (see panels 3 and 4 of Figure 6C for similarly treated cells). Thus, progression past S phase occurring in the absence of cell division leading to hyperploidy is required for nocodazole cytotoxicity in U373MG cells. Hydroxyurea co-treatment also blocked the cytotoxicity of Taxol and vincristine in U373MG cells (Figure 7C). Cytotoxic effects of drugs that function through different mechanisms, e.g. EDTA, were unaffected by hydroxyurea.
Finally, the effect of expressing the G1-specific Cdk inhibitor, p27Kip1, on nocodazole cytotoxicity was determined. Whereas >75% of nocodazole-treated U373MG cells co-infected with Ad-p27 recombinant adenovirus survived, <20% of nocodazole-treated cells co-infected with a control adenovirus expressing prokaryotic ß-galactosidase (Ad-ß-gal) remained viable (Figure 7D). The decline in viability occurring initially after the Ad-p27 infection is probably due to the time it takes to express p27Kip1 protein. Ad-p27 co-infection led to a block in hyperploid peak induction concomitant with the increase of the G2/M peak (Figure 5A), indicating that p27Kip1 blocks nocodazole cytotoxicity through a similar mechanism to hydroxyurea described above. Taken together, the above results suggest that nocodazole, Taxol or vincristine induces lethality in G1-checkpoint-defective U373MG cells by triggering hyperploid progression.
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The effect of blocking hyperploid progression on AMD cytotoxicity was investigated. When S-phase re-entry occurring without cell division leading to hyperploidy was inhibited by hydroxyurea, it abrogated nocodazole cytotoxicity. The cytotoxic effects of currently used anti-cancer drugs, Taxol and vincristine, were also similarly blocked. Ectopic expression of p27Kip1, an inhibitor of G1-specific Cdks, abrogated nocodazole cytotoxicity through a similar mechanism. Thus, the inhibition of S-phase re-entry required for hyperploid progression blocks AMD cytotoxicity against p53-mutated U373MG cells defective in G1 checkpoint. Previously, a similar phenomenon was reported for the bub-1 yeast checkpoint mutant (9).
It appears highly plausible that hyperploid progression induced by Taxol, vincristine or nocodazole will also be abrogated with other Cdk inhibitors besides p27kip1. There are two families of Cdk inhibitors: p21WAF1/Cip1, p27kip1 and p57kip2 belong to one such family with the ability to block all G1-specific Cdks, while p16INK4a, p15INK4b, p18INK4c and p19INK4d belong to another with the capacity to block Cdk4 and Cdk6 specifically (23). The p21 family polypeptides share an N-terminal region required for interacting with G1 cyclin–Cdk complexes. Among them, p27kip1 is thought to represent the most bona fide inhibitor as its level oscillates throughout the cell cycle with the highest level observed during G1 and the lowest during S and G2/M phases. Although not specifically tested, the mechanism of hyperploid progression predicts that inhibiting re-entry into S phase (occurring in the absence of cytokinesis) by these Cdk inhibitors should also block hyperploid progression. Additional experiments are planned to confirm this point.
One possible mechanism through which hyperploid progression may contribute to cell death is via increased expression of genes that sensitize cells to AMD cytotoxicity. The genes encoding the transporters for the uptake of these drugs or the genes whose products mediate cytotoxicity intracellularly may fall into this category. Another possible mechanism is through the increased expression of apoptosis-promoting genes such as Bad and Bax (24). Alternatively, the cell death may be the consequence of inappropriately entering into S or other subsequent cell phases in the absence of cell division. Further experiments are necessary to resolve these points.
Our results suggest that in cells lacking the G1 checkpoint, AMDs induce lethality by triggering hyperploid progression. This provides a unique opportunity to develop a therapy that is specific against tumors lacking the G1 checkpoint. By exploiting the hyperploid-progression-mediated death, a therapy that is tumor specific at the genetic level may be developed. It may have a wide therapeutic applicability as a large fraction of human cancers are mutated in the p53 gene.
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Acknowledgments |
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We thank D.Goodrich of M.D. Anderson Cancer Center and S.Wasserman of University of Texas Southwestern Medical Center for reviewing the manuscript. We thank M.Azzara for preparing viruses and E.Rushing for providing immunofluorescence microscope facility. This work was supported by NIH grant CA60173 and by the Klion Family Fund.
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3 To whom correspondence should be addressed Email: fhong{at}notes.mdacc.tmc.edu
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