Article |
Address correspondence to D.J. Donoghue, Department of Chemistry and Biochemistry, University of California San Diego, Urey Hall, Room 6114, 9500 Gilman Drive, La Jolla, CA 92093-0367. Tel.: (858) 534-2167. Fax: (858) 534-7481. E-mail: ddonoghue{at}ucsd.edu
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Abstract |
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Key Words: Speedy; siRNA; proliferation; restriction point; G1/S
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Introduction |
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Because cdk2 is central in controlling cell proliferation, it is subject to many levels of regulation. Although cdk2 is expressed relatively ubiquitously throughout the cell cycle, it is highly regulated by a series of activating and inactivating phosphorylations (Ekholm and Reed, 2000). Additionally, cdk2 activity is limited by the availability of a cyclin binding partner. Cyclin proteins are regulated at the protein expression level, being synthesized and destroyed in an oscillating manner throughout the cell cycle, thereby assuring a narrow window of cdk activation (Ekholm and Reed, 2000). In mammalian cells, cdk2 forms active complexes with both cyclin E and cyclin A (Fang and Newport, 1991; for review see Ekholm and Reed, 2000). Crystallography studies demonstrate that cyclin binding allows for structural modifications within the catalytic cleft of the cdk molecule, enhancing cdk access to ATP and relative substrates (Jeffrey et al., 1995). Furthermore, both the formation and the activity of the cdkcyclin complex are subject to inhibition by small inhibitory proteins (cdk inhibitors [CKIs]; Pines, 1994). Although much is known about the regulatory mechanisms governing cdk activation, questions still remain regarding the precise timing and manner by which all of these regulatory mechanisms function, particularly during times of cellular stress, DNA damage, and oncogenesis.
Previously, our laboratory reported the identification of a novel Xenopus cell cycle regulatory gene, X-Spy1 (Lenormand et al., 1999). Shortly after our publication of X-Spy1, Ferby et al. (1999) reported the identification of a Xenopus gene, p33ringo, which is 90% homologous to X-Spy1. Microinjection of either X-Spy1 or p33ringo mRNA into Xenopus oocytes elicits rapid maturation in the absence of hormone. Additionally, X-Spy1 and p33ringo are essential for progesterone-stimulated maturation of Xenopus oocytes (Ferby et al., 1999; Lenormand et al., 1999). Moreover, X-Spy1 and p33ringo appear to be functionally redundant in the maturation process, as inhibiting only one of these proteins at a time with antisense oligonucleotides does not prevent maturation (Ferby et al., 1999). Interestingly, in Xenopus oocytes, X-Spy1 and p33ringo were both found to bind and prematurely activate cdk2 in a p21-independent manner (Ferby et al., 1999; Lenormand et al., 1999). Recently, it has been reported that activation of cdk2 by p33ringo does not rely on the phosphorylation of Thr161, suggesting a novel mechanism for cdk2 activation (Karaiskou et al., 2001). Thus, X-Spy1 and p33ringo represent a new class of cell cycle regulatory proteins that directly interact with and activate cdk2, independent of classical regulatory mechanisms.
Here we report the identification and characterization of the human homologue of Speedy (Spy1). Similar to its Xenopus counterpart, human Speedy is able to induce maturation in Xenopus oocytes upon microinjection, suggesting a role in cell cycle progression. Human Speedy mRNA is present in a range of normal tissues and immortalized cell lines and is regulated in a cell cycledependent manner. We demonstrate that Spy1 interacts with human cdk2 independent of cyclin binding and stimulates its kinase activity in mammalian cells. Furthermore, Spy1 overexpression in various cell lines rapidly induces cell cycle progression, an action that is dependent on cdk2 activation. Importantly, we show that depleting endogenous Spy1 significantly decreases cell proliferation, demonstrating a physiological function for Spy1. Thus, human Speedy is a novel, essential inducer of cell cycle progression that functions through a unique pathway of cdk2 activation.
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Results |
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Speedy mRNA is expressed in a variety of human tissues and cell lines
To determine if Speedy mRNA is endogenously expressed in a variety of normal human tissues and immortalized cell lines, reverse transcriptase PCR (RT-PCR) was performed on mRNA isolated from 15 different human tissues as well as 3 different cell lines. Primers were used that are specific for the conserved region of human Speedy. As shown in Fig. 3 A, Spy1 mRNA is expressed at high levels in human fetal brain (lane 6) and the thyroid gland (lane 12). Significant amounts of Spy1 mRNA are also detected in human thymus (lane 3), salivary gland (lane 4), liver (lane 5), cerebellum (lane 13), heart (lane 14), and placenta (lane 15). As shown in Fig. 3 C, Spy1 mRNA is present in the human teratocarcinoma cell line Ntera-2 and the human embryonic kidney 293T cell line (lanes 3 and 4, respectively); yet Spy1 mRNA was not detected in the human epitheloid carcinoma cell line HeLa (lane 6). Notably, when mRNA levels for HeLa cells are increased 10-fold, Spy1 RNA can be detected (unpublished data). As a control, Fig. 3, B and D, demonstrates that the total mRNA used was of equal quality and quantity. PCR products were sequenced to confirm that the 360-bp band was indeed Speedy (unpublished data). Smaller bands did not have informative sequences and are likely degradation products of Spy1. Thus, Spy1 mRNA is expressed in both a variety of immortalized cell lines as well as in several human tissue types.
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Spy1 is localized in the nucleus and interacts with human cdk2
To determine the subcellular localization of Speedy, a myc-tagged human Speedy construct (mycSpy1) was engineered. COS-1 cells were transiently transfected with mycSpy1 or an empty vector as a control. Indirect immunofluorescence was then performed to examine the subcellular localization of Spy1. Treatment of cells with the c-myc (9E10) antibody showed that mycSpy1 was expressed exclusively in the nucleus (Fig. 4 A, bottom left). Hoechst dye was used to stain the nuclei of all cells (Fig. 4 A, right).
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We then wished to examine the kinase activity of cdk2. Lysates from mock- or mycSpy1-transfected 293T cells were immunoprecipitated with antibodies against cdk2 and subjected to an in vitro histone H1 phosphorylation assay. Speedy-expressing cells had a significant increase in cdk2 kinase activity (Fig. 4 C, top). As a control, the protein levels of cdk2 were examined and found to be present in equivalent levels (Fig. 4 C, bottom). Although H1-associated kinase activity is widely used and highly indicative of the relative levels of kinase activity, the actual alteration in cdk2 activity on in vivo substrates may differ. These results indicate, consistent with prior observations in Xenopus oocytes, that human Spy1 binds to cdk2 and stimulates cdk2 kinase activity.
Interestingly, we have been unable to show consistent binding of Spy1 with any of the cyclins (unpublished data). Furthermore, when lysates from mock- or mycSpy1-transfected cells were immunoprecipitated with antibodies against cdc2 and subjected to an in vitro histone H1 phosphorylation assay, we found no significant increase in cdc2 kinase activity in Spy1-expressing cells (Fig. 4 D, top) that have equal levels of cdc2 protein (Fig. 4 D, bottom).
Spy1 enhances cell proliferation in mammalian cells
To determine if Spy1 plays a role in regulating the mammalian cell cycle, growth curves were obtained comparing control-transfected cells against cells expressing mycSpy1. Transiently transfected HeLa, NIH3T3, and Ntera-2 cells were counted every 24 h after transfection for 96 h. As shown in Fig. 5, AC, cells overexpressing Spy1 grew significantly faster than mock cells. Trypan blue exclusion was used to distinguish between live and dead cells and no appreciable difference was noted in cell death (unpublished data). These data indicate that Spy1 expression enhances overall cell growth rate.
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Spy1-enhanced cell proliferation is dependent on cdk2 kinase activity
To determine if Spy1 requires cdk2 kinase activity in order to enhance cell proliferation, we used a dominant-negative cdk2, cdk2D145N (van den Heuvel and Harlow, 1993). We additionally exploited a drug known to selectively inhibit cdk2 at low doses, olomoucine (Vesely et al., 1994). The dose of olomoucine used, 7 µM, is the IC50 value for cdk2 and cdc2 inhibition. This dose has no effect on any other kinase tested including both cdk4 and cdk6 (Vesely et al., 1994). Fig. 7 A demonstrates that Spy1 is unable to stimulate proliferation of cells where cdk2 kinase activity has been inhibited either through the use of the dominant-negative cdk2 or olomoucine. This is not due to an alteration in the expression of mycSpy1 protein (Fig. 7 B).
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Discussion |
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Previous studies demonstrated that the Xenopus Spy1 homologues, X-Spy1 and p33ringo, bind to and activate cdk2 (Ferby et al., 1999; Lenormand et al., 1999). Moreover, p33ringo was shown to bind to and activate cdc2 (Ferby et al., 1999). In these studies, X-Spy1 and p33ringo are associated with rapid oocyte maturation, demonstrating the ability of X-Spy1 to progress cells through the G2/M transition of the cell cycle. Similar to these reports, we have found that human Speedy activates cdk2 in mammalian cells. Yet contrary to the Xenopus system, Spy1 in mammalian cells is associated with rapid progression of cells through G1/S. Furthermore, under our experimental conditions, we have been unable to demonstrate any direct binding of Spy1 with cdc2 or any enhanced activity of cdc2 in Spy1-expressing cells. We do, however, demonstrate that Spy1-expressing cells exhibit enhanced expression of phospho-histone H3, an indicator of cell division, and thus cannot rule out that Spy1 may exert some as yet undetermined effects in G2/M phase of the cell cycle. The apparent discrepancies between results obtained with Xenopus p33ringo (Ferby et al., 1999; Karaiskou et al., 2001) and human Spy1 may possibly be explained by species differences in the regulatory properties of the Spy1 protein itself. It is evident from this report that Spy1 mRNA is detectable primarily during G1/S phase of the cell cycle in mammalian cells. Additionally, our laboratory has preliminary data suggesting that Spy1 protein is degraded during G2 phase of the cell cycle (unpublished data). Xenopus studies, on the other hand, indicate that X-Spy1 and p33ringo protein exists at the G2/M border (Ferby et al., 1999; Lenormand et al., 1999). The fact that Spy1 is differently regulated between species is interesting and merits further investigation.
The possibility that Spy1 has biological function at various phases of the cell cycle depending on its production and destruction is itself interesting and reflects a characteristic common to the growing family of cyclin proteins. Furthermore, we demonstrate here that Spy1 mRNA is expressed in a cell cycledependent manner, similar to cyclin expression profiles. Yet, at the amino acid level, Spy1 is not significantly related to the archetypical cyclins. Future experiments determining the mechanism by which Spy1 activates cdk2 will reveal if Spy1 is indeed a potential cyclin-like protein. We cannot yet rule out that Spy1 is binding cdk2 via a complex containing other proteins. Perhaps Spy1 functions by altering the binding of cell cycle inhibitors to cdks.
Recently p33ringo was demonstrated to activate both cdc2 and cdk2 regardless of the phosphorylation status of Thr160 and Thr161, respectively, and in the absence of cyclin binding (Karaiskou et al., 2001). This implies that X-Spy1 and p33ringo activate their relevant cdk via a unique mechanism. Interestingly, we have been unable to observe any cyclin binding to Spy1 via coimmunoprecipitation (unpublished data). This suggests that Spy1, like p33ringo, activates cdk2 via a unique pathway that is independent of cyclin binding. Consistent with this, depletion of Spy1 mRNA slows cell growth considerably, yet cells are still able to replicate and divide. Additionally, Spy1 mRNA is found at vastly different levels in select tissues, suggesting that Spy1-mediated cdk2 activation will have unique tissue-specific functions. It will be interesting to further determine how Spy1 binds and activates cdk2 and to determine the effects of Spy1 on cdk2cyclin E/cyclin A complex formation. It is becoming increasingly evident that there are multiple pathways leading to cdk2 activation, ultimately promoting entry into S phase. The most well-established pathway leading to S phase is the RB pathway, comprising RB protein along with its upstream regulators cyclin D and cdk4/6 and pRB-regulated E2F transcription factors (Bartek et al., 1996). Accumulating evidence supports that the proto-oncogene Myc functions in parallel to RB in a pathway that is independent of E2F activation (Santoni-Rugiu et al., 2000). It will be interesting to determine if Spy1 is a critical component of one of these pathways regulating the onset of DNA replication. Moreover, it will be important to address if Spy1cdk2 binding is altered during times of cellular stress or DNA damage, because Spy1 was initially isolated by its ability to rescue a Rad1-deficient strain of Schizosaccharomyces pombe from UV damage.
In summary, the results presented here characterize a novel human cell cycle gene that plays a role in the progression of mammalian cells through its ability to bind and activate cdk2. We have established that endogenous Spy1 is essential for the proliferation of mammalian cell lines and that Spy1 is expressed over a wide range of human tissues, opening up the possibility that this gene may be involved in human cell cycle disorders such as oncogenesis. There are numerous questions remaining regarding the regulation of Spy1; the answers of which may provide valuable insight into the mechanics of the mammalian cell cycle.
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Materials and methods |
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Plasmid construction and mRNA isolation
Human Speedy was isolated from a testis library in a TriplEx vector (CLONTECH Laboratories Inc.). pTriplEx was excised from
TriplEx per the manufacturer's instructions. EcoRI and SacI sites were used to move human Spy1 from pTriplEx to pSB5, a modified version of pSP64(polyA) (Promega) containing EcoRI in the multicloning region with XhoI instead of EcoRI as the linearization site. To generate the mycSpy1 clone, a NdeI site that incorporates the start codon of human Speedy was added via QuikChange site-directed mutagenesis (Stratagene). A pair of oligonucleotides (D1904/D1905) was inserted into the multicloning region of the pCS3 + MT vector at the BglII site, inserting a NdeI site in which the ATG was in frame with the myc tag coding region. Then Spy1 was ligated into pCS3 + MT via a three-part ligation using HindIII, NdeI, and XbaI sites to construct mycSpy1. The X-Spy1 gene in the pSP64 vector was constructed as described previously (Lenormand et al., 1999). The deletion mutants of X-Spy1 were constructed by first inserting two silent restriction sites into X-Spy1 via QuikChange site-directed mutagenesis (Stratagene). A pair of oligonucleotides (D2423/D2424) inserted a NcoI silent site at aa 94 of X-Spy1 and a second pair of oligonucleotides (D2433/D2434) inserted a XhoI silent site at aa 137 of X-Spy1 in pSP64(poly A). Subsequently, X-Spy1 was cut with either NdeI/NcoI, NcoI/XhoI, or XhoI/BamHI, and in each case an
100-aa piece of X-Spy1 was removed. Then
50 aa were added back, via a pair of complementary oligonucleotides, in frame to create the deletion mutants lacking
50 aa each. The pairs of oligonucleotides used were as follows: D2426/2427 for
1; D2428/D2429 for
2; D2435/D2436 for
3; D2437/D2438 for
4; D2439/D2440 for
5; and D2441/D2442 for
6. mRNAs were synthesized from pSP64 constructs using SP6 polymerase as described previously (Freeman et al., 1989).
The dominant-negative cdk2 vector (pCMV-cdk2D145N) was a gift from Ed Harlow and Sander van den Heuvel (Massachusetts General Hospital Cancer Center, Charlestown, MA), and has been previously described (van den Heuvel and Harlow, 1993).
Oocyte microinjections
Stage VI oocytes were dissected manually in modified Barth salineHepes (MBS-H) buffer. They were then either treated with progesterone (30 µM), microinjected with 50 nl of RNA at 1.0 mg/ml, or microinjected with 50 nl of diethylpyrocarbonate (DEPC)-treated H2O and scored for GVBD as indicated by the appearance of a white spot on the animal pole, as described previously (Freeman et al., 1989).
Cell culture and synchronization
293T cells are a human embryonic kidney cell line (American Type Culture Collection [ATCC]), Ntera-2 cells are a human teratocarcinoma cell line (ATCC), HeLa cells are a human epitheloid carcinoma cell line (ATCC), COS-1 cells are an SV40-transformed African green monkey kidney cell line (ATCC), and NIH3T3 cells are an embryonic, contact inhibited, NIH Swiss mouse cell line (ATCC). All of these cell lines were maintained in DME (GIBCO BRL) supplemented with 0.1% penicillinstreptomycin (Sigma-Aldrich) and 10% FBS. Ntera-2 cells were also supplemented with 4 mM L-glutamine (Sigma-Aldrich). Cells were incubated at 37°C in 10% (293T, COS-1, and Ntera-2 cells) or 5% CO2 (HeLa and NIH3T3 cells).
Double thymidine block was used to synchronize the cell cycle. 293T cells were first treated with 100 mM thymidine in DME for 19 h at 37°C in 10% CO2. Cells were then washed with Tris-saline and incubated in fresh media for 9 h. Thymidine/DME was then added for the second thymidine block, and cells were incubated for 17 h. The final Tris-saline wash and incubation in fresh media was followed by the beginning of time points, as cells exit simultaneously from Go of the cell cycle.
Human Spy1 antibody isolation
The human Spy1 antibody was produced in collaboration with Pocono Rabbit Farm and Laboratory. Rabbits were immunized with KLH-conjugated peptides against aa 251263 of human Spy1. ELISAs were performed using BSA-conjugated peptide, and further boosts were done every 2 wk with the KLH-conjugated peptide. Specificity of the antibody was determined using peptide-blocked antibody and preimmune serum derived from the same rabbit. The human Spy1 antibody specifically detects an endogenous protein of 34 kD.
RT-PCR
Total RNA from 15 different human tissues was obtained from the Human Total RNA Master Panel II (CLONTECH Laboratories, Inc.). Total RNA was prepared from cells (3 x 106) using RNeasy (QIAGEN) per the manufacturer's instructions. The quantity and quality of mRNA were identified by running samples on a 1.2% agarose formaldehyde gel. 1 µg of RNA was reverse transcribed with 2.5 U of AMV reverse transcriptase following the supplier's suggestions (Promega). The reverse transcriptase reactant was mixed in a final volume of 20 µl together with 50 pmol of primer pairs and 25 µM of each deoxynucleoside triphosphate (dNTP) and amplified with 2.5 U of Tf1 DNA polymerase (Promega) for 30 cycles (denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 68°C for 1 min). This number of cycles was previously determined to be optimal for detecting the signal in a linear range. The primer sequences used were 5'-ATT GGG AAA CCA AAA TGA GGC-3' (sense) and 5'-TCC TGG TAT GCT CAC TTA TAG-3' (antisense). Positive control RNA and primers were included with the Access RT-PCR System (Promega) and used per the manufacturer's instructions. Amplification products were visualized and photographed using TAE/EtBr gel electrophoresis. Products were then gel purified using GeneClean (Bio101), following the manufacturer's instructions, and were cloned using the pGEM-T Easy Vector System. Each of the cloned products was then sequenced (UCSD Cancer Center DNA Sequencing Service).
Immunofluorescence microscopy
293T or COS-1 cells were plated onto glass coverslips (collagen coated for 293T cells) and cultured as described above. Cells were transiently transfected by calcium phosphate precipitation (Chen and Okayama, 1987) with 5 µg of mycSpy1 in a 60-mm dish. 3648 h after transfection, cells were fixed with 3% paraformaldehyde in PBS for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. The 9E10 monoclonal myc antiserum (Santa Cruz Biotechnology, Inc.) was used to detect mycSpy1 with FITC-conjugated goat antimouse secondary antibody (Boehringer).
For M phase marker assay (phospho-histone H3), 293T cells were incubated in blocking solution (0.1% Triton-X, 1.5% glycine, 2.5% FBS in PBS) for 30 min at room temperature (RT). An antisera that specifically recognizes the phosphorylated form of histone H3 (Upstate Biotechnology) was added at a 1:200 dilution in blocking solution for 1 h at RT. FITC-conjugated antimouse was then used at a 1:200 dilution.
For S phase marker assay (BrdU), 293T cells were treated as described above. In addition, 100 µl of 6 mg/ml BrdU labeling solution in PBS was added on the second day after transfection for 1 h at 37°C in 10% CO2. Anti-BrdU (Becton Dickinson) was added to coverslips at a 1:50 dilution in blocking solution for 1 h at RT. FITC-conjugated antimouse was used at a dilution of 1:150 for visualization.
Immunoprecipitations and immunoblotting
Subconfluent 293T cells were transfected with 5 µg of DNA by calcium phosphate precipitation in a 10-cm dish. 24 h later, cells were harvested and lysed in 0.1% NP-40 lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 0.1% NP-40, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, 1 mM DTT, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, 10 µg/ml leupeptin).
For immunoprecipitation, cell lysates were clarified with protein ASepharose beads (Sigma-Aldrich) and subsequently incubated with primary antisera (myc9E10, cdk2-D12, cdk2-M2, cdc2-p34 [17]; Santa Cruz Biotechnology, Inc.) as indicated overnight at 4°C, followed by the addition of protein ASepharose and incubation at 4°C with gentle rotation for an additional 1 h. These complexes were then washed extensively with 0.1% NP-40 lysis buffer and resolved by 10% SDS-PAGE. The membrane was immunoblotted with the indicated antisera followed by ECL (Amersham Pharmacia Biotech). The histone H1 kinase assays were performed as previously described (Kong et al., 2000). siRNA-treated cells were serum starved using media containing 0.2% FBS for 12 h before harvesting for histone kinase assays.
Cell proliferation assays
Cell growth curves.
HeLa, NIH3T3, and Ntera-2 cells were seeded at a density of 105 cells/plate (10 cm) and were transfected in triplicate 24 h later with 5 µg of either empty vector or mycSpy1. Every 24 h after transfection, three plates of both mycSpy1 or empty vector control were trypsinized and counted by trypan blue exclusion. The average values of three plates are represented (±SEM). Coverslips were placed on each plate to assess transfection efficiency by indirect immunofluorescence. HeLa cells transfected with 40% efficiency, Ntera-2 cells with 45% efficiency, and NIH3T3 cells with 30% efficiency. Remaining cells for that time point were pooled, lysed, subjected to SDS-PAGE, and immunoblotted for mycSpy1 expression. This experiment was repeated three times.
293T cells were seeded at a density of 5 x 104 cells/plate (60 mm) and were transfected 24 h later with 2.5 µg of either empty vector or mycSpy1. 293T cells demonstrated a transfection efficiency of 50%, as assessed by immunofluorescence. Cells were trypsinized and counted every 24 h after transfection (day 1). The average values of three plates are represented (±SEM). Cell lysate samples from day 4 were analyzed for mycSpy1 expression by immunoblotting.
Tetrazolium assay.
293T cells, transfected as described above, were split onto a 96-well plate. 20 µl of MTT (Sigma-Aldrich) stock (5 mg/ml in PBS) was added to each and incubated at 37°C in 10% CO2 for 2 h. Then 100 µl of extraction buffer (20% SDS in DMF/H2O, 2.5% of 80% acetic acid, 2.5% 1 M HCl, pH 4.7) was added to each well overnight with incubation at 37°C in 10% CO2. Cells were then pulled at the indicated times and the absorbances were taken at 570 nm.
Inhibition of cdk2 growth curves.
293T cells were seeded at a density of 4 x 104 cells/plate (60 mm) and were transfected 24 h later with 5 µg of total DNA. Combinations of empty vector control and mycSpy1 were used with or without cdk2D145N. Furthermore, mycSpy1- or control-transfected cells were either treated with DMSO control or 7 µM olomoucine (Sigma-Aldrich). Cells were trypsinized and triplicate plates were counted every 24 h for each condition. The average values are represented (±SEM). Remaining cells at each time point were lysed, subjected to SDS-PAGE, and analyzed for mycSpy1 expression by immunoblotting.
Flow cytometry
293T cells were transfected as described above with 10 µg of DNA. Cells were trypsinized at the indicated times, washed twice in PBS, resuspended at 2 x 106 cells in 1 ml of PBS, fixed by the addition of an equal amount of ethanol, and frozen. Within 1 wk, fixed cells were pelleted, washed, and resuspended in 300 µl of PBS. Samples were then treated with 1 µl of 10 mg/ml stock of DNase free RNase (Promega) and 50 µl of 500 mg/ml propidium iodide (Boehringer) stock solution. Data were collected on a flow cytometer within 6 h. The multicycle (M-cycle) program for cell cycle distribution histograms (Phoenix Flow Systems, Inc.) was used to analyze the data.
siRNA
RNA interference assays were conducted through the construction of siRNA for Spy1 (siSpy). The siSpy oligo (-GTACGAAATTTTTCCATGG-) was synthesized, purified, and duplexed by Dharmacon Research. As a negative control, a luciferase GL2 duplex siRNA was used (D-1000-Lu-5; Dharmacon Research).
siRNA transfection.
Subconfluent 293T cells (105 cells/ml) were seeded in 10-cm dishes. Cells were transfected using calcium phosphate precipitation with 0.6 nM siSpy or siluc-GL2 duplex per plate. Cells were incubated at 3% CO2 for 12 h and then returned to 10% CO2 after changing the media.
siRNA growth curves.
Every 24 h, plates were pulled in triplicate and both live and dead cells were counted by trypan blue exclusion. Surviving cells were averaged at each time point and their SEM was determined. Remaining cells were pooled for mRNA preparation as described above and subjected to RT-PCR analysis for Spy1 mRNA production. The quantity and quality of mRNA was determined through formaldehyde gel electrophoresis.
Mitotic index.
293T cells were plated on collagen-coated coverslips and transfected with siSpy or siluc-GL2 control as described above. After the media change, cells were grown for 48 h, stained with Hoechst stain (0.7% NP-40, 4.7% formaldehyde, 11 µg/ml Hoechst stain in PBS), and analyzed by fluorescence microscopy.
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Footnotes |
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J.-L. Lenormand's present address is Institut de Biologie Structurale, 41 Rue Horowitz, 38027 Grenoble Cedex, France.
R.W. Dellinger's present address is NewBiotics, Inc., 11760 Sorrento Valley Road, Suite E, San Diego, CA 92121.
* Abbreviations used in this paper: aa, amino acid(s); ATCC, American Type Culture Collection; cdk, cyclin-dependent kinase; GVBD, germinal vesicle breakdown; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; RB, retinoblastoma; RT, room temperature; RT-PCR, reverse transcriptase PCR; siRNA, small interference RNA
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Acknowledgments |
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This work is supported by a grant from the National Institutes of Health (NIH) to D.J. Donoghue (RO1-CA34456). E.A. Barnes acknowledges support from an NIH National Research Service Award grant (T32-CA09523). L.A. Porter acknowledges support from the Pete Lopiccola Fellowship in Cancer Research.
Submitted: 17 September 2001
Revised: 15 March 2002
Accepted: 19 March 2002
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