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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 1039-1046
NEOPLASIA
From the Department of Adult Oncology, Dana-Farber Cancer Institute,
and the Department of Medicine, Harvard Medical School, Boston, MA; the
Department of Hematology, Singapore General Hospital, Republic of
Singapore; the Department of Pediatrics, Jikei University School of
Medicine, Tokyo; and the Center for Molecular Biology and Cytogenetics,
SRL, Inc, Tokyo, Japan.
It has been reported that the activation of multiple myeloma (MM)
cells by CD40 induces proliferation, growth arrest, and apoptosis. To
determine whether the biologic sequelae of CD40 activation in MM
cells depends on p53 function, we identified temperature-sensitive p53 mutations in the RPMI 8226 (tsp53E285K) and the HS Sultan (tsp53Y163H) MM cell lines. These cells were then
used as a model system of inducible wtp53-like function because wild-type-like p53 is induced at permissive (30°C) but not at restrictive (37°C) temperatures. Using p21-luciferase reporter assays, we confirmed that CD40 induces p53 transactivation in RPMI 8226 and HS Sultan cells cultured under permissive, but not restrictive,
conditions. Furthermore, CD40 activation of these MM cells under
permissive, but not restrictive, temperatures increased the expression
of p53 and p21 mRNA and protein. Importantly, CD40 activation induced
the proliferation of RPMI 8226 and HS Sultan cells at restrictive
temperatures and growth arrest and increased subG1 phase cells at
permissive temperatures. These data confirmed that CD40 activation
might have distinct biologic sequelae in MM cells, depending on their
p53 status.
(Blood. 2000;95:1039-1046)
Others1-6 and we have demonstrated the
effect of CD40 activation on the proliferation and differentiation of B
lymphocytes. Triggering of multiple myeloma (MM) cells by cell surface
CD40 has been shown to induce the proliferation of tumor cells in some studies7-12 and to trigger growth arrest13 and
apoptosis14 in others. Ligation of CD40 on MM cells also
induces the secretion of transforming growth factor- Earlier studies characterize the signaling cascades mediating
IL-6-triggered growth21,22 and survival23-25 in
MM cells and demonstrate that apoptosis of MM cells, induced by
wild-type p53 (wtp53)26,27 and p21,28 can be
abrogated by treatment with exogenous IL-6. These studies establish
IL-6 as a major autocrine and paracrine growth factor, especially in
CD40-activated MM cells. Cell cycle regulatory proteins, including
wtp53, directly regulate IL-6 secretion; wtp53 represses the IL-6
promoter, whereas IL-6 promoter activity is not down-regulated by
mtp53.29-31 These data suggest that the CD40 activation of
MM cells may induce growth and survival rather than growth arrest and
apoptosis, depending on their p53 status. In addition, they suggest
that the activation of CD40 can alter the expression of p53 and
consequently of p21 proteins. However, the direct role of CD40
activation in regulating p53 and p21 expression and p53-dependent cell
cycle control has not been studied.
In this study, we first identified temperature-sensitive
missense mutations in the p53 gene in 2 human MM-derived cell lines: codon 285, Glu [GAG] to Lys [AAG] in RPMI 8226, and codon 163, Tyr
[TAC] to His [CAC] in HS Sultan MM cells. These cell lines were
then used as a model system of inducible wtp53-like function because
wt-like p53 conformation is induced in these cells at permissive,
but not at restrictive, temperatures. Using p21-luciferase reporter
assays, we confirmed that CD40 activation of these MM cell lines at
permissive, but not at restrictive, temperatures increased p53
transactivation activity. We then showed that p21 and p53 mRNA and
protein expression were increased in CD40-activated RPMI 8226 and
HS Sultan cells cultured under permissive, but not restrictive,
conditions. Finally, CD40 activation of RPMI 8226 and HS Sultan cells
at restrictive temperatures increased G1/S transition and tumor cell
proliferation; in contrast, CD40 activation of these cells at
permissive temperatures increased growth arrest and subG1 phase cells.
These data demonstrated that CD40 activation mediated p53-dependent
cell cycle regulation in human MM cell lines.
Cell lines and transfectants
Cell proliferation and apoptosis assays
Cell cycle distribution analysis RPMI 8226 and HS Sultan cells (0.5 × 106 cells/mL) were cultured in media with formalin-fixed NIH3T3/CD40LT, NIH3T3/wt, or NIH3T3/vt for up to 144 hours. Cells were fixed and permeabilized with 80% ethanol on ice for 1 hour and resuspended in phosphate-buffered saline (PBS) with DNase-free RNase (10 µg/mL; Boehringer Mannheim, Indianapolis, IN) and stained with propidium iodide (PI) (15 µg/mL, Sigma Diagnostics). Cell cycle distribution was assessed by flow cytometric analysis on the Coulter Epics XL flow cytometer (Coulter, Hialeah, FL).Antibodies for immunoprecipitation and Western blotting The following antibodies (Abs) were used: Ab-5 anti-wtp53 (clone PAb 1620) monoclonal Ab (mAb), which recognizes wtp53 conformation; Ab-3 anti-mtp53 (clone PAb 240) mAb, which recognizes mtp53 conformation; and Ab-1 anti-actin mAb (all from Oncogene Science, Cambridge, MA); DO-1 horseradish peroxidase (HRP)-conjugated anti-pantropic (ptp53) mAb, C-19 goat anti-p21 polyclonal Ab (pAb), HRP-conjugated anti-mouse immunoglobulin (Ig), and HRP-conjugated donkey anti-goat IgG mAb (all from Santa Cruz Biotechnology, Santa Cruz, CA); and DM 1A antitubulin mAb (Sigma Diagnostics).Immunoprecipitation and Western blotting Total cell lysates were obtained as previously described15,32 and quantified by Bradford's method (Bio-Rad, Hercules, CA). For immunoprecipitation, 1 mL cell lysate (1 mg/mL) was incubated with 10 µg of the relevant Ab and 100 µL of 10% Protein-A Sepharose CL-4B (Pharmacia Biotech, Uppsala, Sweden); for Western blotting, total protein (30 µg/sample) was used. The samples were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), blocked with 5% skim milk (Saco Foods, Middleton, WI) in PBS, 0.2% Tween-20, and 0.02% NaN3 (both from Sigma Diagnostics); and immunoblotted with 10 µg relevant Ab, followed by appropriate HRP-conjugated secondary Ab. Detection was performed by HRP chemiluminescence (ECL; Amersham Life Science) autoradiography (Biomax MR; Eastman Kodak, Rochester, NY). The relative intensity of expression was assessed using imaging densitometry (AlphaImager 2000; Alpha Innotech, San Leandro, CA).Direct DNA sequencing of human p53 gene exons IV to IX Genomic DNA was extracted from both MM cell lines (2 × 106/sample) using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) and was quantified (260 nm) on the DU 640 Spectrophotometer (Beckman Instruments, Columbia, MD). In the first-step polymerase chain reaction (PCR), a 2.8-kb fragment of DNA that encodes exons IV through IX was obtained using the LA-PCR Kit (Takara, Tokyo, Japan). The PCR conditions were as follows: forward primer, 5'-AGGACCTGGTCCTCTGACTG-3'; reverse primer, 5'-TAGACTGGAAACTTCCACTTG-3'; 94°C at 30 seconds (denaturation); 58°C at 60 seconds (annealing); and 72°C at 60 seconds (extension) for 35 cycles. Single-strand DNA33 was produced by asymmetrical PCR using the deoxy chain termination method with end-labeled sequencing primers for exon IV (forward primer, 5'-TTTCACCCATCTACAGTCCCC-3'; reverse primer, 5'-GAAGTCTCATGGAAGCCAGCC-3'); exons V and VI (forward primer, 5'-TTCCTCTTCTACAGTACTCC-3'; reverse primer, 5'-AGTTGCAAACCAGACCTCAGG-3'); exon VII (forward primer, 5'-CCAAGGCGCACTGGCCTCATC-3'; reverse primer, 5'-TCAGCGGCAAGCAGAGGCTGG-3'); and exons VIII and IX (forward primer, 5'-CCTATCCTGAGTAGTGGTAAT-3'; reverse primer, 5'-TAAGAGGTCCCAAGACTTAGT-3') of the human p53 gene (PRISM DyeDeoxy Terminator Sequencing Kit FS; Applied Biosystems, Foster City, CA). DNA sequencing was performed on the ABIPRISM 310 Genetic Analyzer (Applied Biosystems). Sequencing of the antisense DNA was used to confirm mutations.p21-Luciferase reporter assay p21-Luciferase reporter plasmid either PG13-luc, which contains 13 copies of a normal p53-binding site, or MG15-luc, which contains 15 copies of a subtly mutated p53-binding site (both kind gifts of Dr Bert
Vogelstein, Johns Hopkins University, Baltimore, MD)34was
transfected (10 µg/sample) into RPMI 8226 and HS Sultan MM cell lines
(10 × 106/sample) using electroporation (Bio-Rad
Gene Pulser; Bio-Rad). Tumor cells were incubated at 37°C for 14 hours to 16 hours and then cultured in media or with formalin-fixed
NIH3T3/CD40LT for up to 40 hours. Next MM cells were harvested, and
luciferase activity was expressed as relative light units (Luciferase
Assay System; Promega) measured using the Monolight 2010 Luminometer
(Analytical Luminescence Laboratory, Frederick, MD). Total protein
content was used for the normalization of luciferase activity.
Experiments were performed in triplicate, and results were expressed as
mean ± SEM normalized luciferase activity.
Ribonuclease protection assay RPMI 8226 or HS Sultan MM cells (10 × 106/sample) were cultured at restrictive (37°C) and permissive (30°C) temperatures in media and with formalin-fixed NIH3T3/CD40LT, NIH3T3/wt, or NIH3T3/vt for up to 72 hours. Total cellular RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) and quantified (260 nm) on the DU 640 Spectrophotometer (Beckman Instruments). The following cDNA templates were used: p21 cDNA cloned to pcDNA3 plasmid (Invitrogen, Carlsbad, CA) and linearized at the Kpn I1164 restriction site (Gibco BRL); p53 cDNA cloned to pcDNA3 plasmid (Invitrogen) and linearized at the BssS I1019 restriction site (New England Biolabs, Beverly, MA); and pTri-actin control cDNA (Ambion, Austin, TX). Next -32P-UTP (Dupont NEN)-labeled antisense riboprobes were
transcribed using the SP6 RNA polymerase promoter from these cDNA
templates (MAXIscript T7/SP6 in vitro transcription kit; Ambion) and
purified on a 5% polyacrylamide/8 mol/L urea sequencing gel.
Ribonuclease protection assay (RPA) (10 µg total RNA/sample) was
performed using the RPA III kit (Ambion), resolved on a denaturing 5%
polyacrylamide/8 mol/L urea sequencing gel, and detected by
autoradiography (Eastman Kodak). The relative intensity of expression
was assessed by imaging densitometry (AlphaImager 2000; Alpha Innotech).
Characterization of p53 mutations in RPMI 8226 and HS Sultan MM cell lines To determine the molecular mechanisms for the contrasting biologic sequelae of CD40 activation in MM cells, we first characterized the p53 gene in 2 human MM cell lines, RPMI 8226 and HS Sultan. Polymerase chain reaction and direct DNA sequencing identified a previously described temperature-sensitive missense mutation (tsp53E285K, codon 285 (Glu [GAG] to Lys [AAG])) capable of producing p53 transactivation function at 30°C in RPMI 8226 cells 35 and another missense mutation (mtp53Y163H, codon 163 (Tyr [TAC] to His [CAC])) in HS Sultan cells. Sequencing of the antisense DNA was used to confirm mutations.Characterization of temperature-dependent p53 protein conformation in RPMI 8226 and HS Sultan MM cell lines Because missense mutations in the central region (codons 101-318) of the human p53 gene often confer temperature sensitivity (tsp53),36 we next characterized the effect of temperature on the expression of p53 protein using conformation-specific mAbs directed against wtp53 (PAb 1620) and mtp53 (PAb 240) for immunoprecipitation, followed by immunoblotting with a nonconformation-specific pantropic p53 (ptp53) mAb (DO-1) (Figure 1). Specifically, the effect of temperature on wtp53 expression and mtp53 conformation was assessed by culturing tumor cells at restrictive (37°C) rather than permissive (30°C) temperatures. RPMI 8226 and HS Sultan cells predominantly express mtp53 when cultured at restrictive temperatures (Figure 1A), and they express wtp53 when cultured at permissive temperatures (Figure 1B). To confirm the above pattern of wtp53 expression in these MM cells, we also carried out mock- and -irradiated cultures of these tumor cells at
37°C or 30°C and assayed for p53 protein expression as
described above. Expression of wtp53 was increased by -irradiation
in RPMI 8226 and HS Sultan cells cultured at permissive (Figure 1D),
but not at restrictive (Figure 1C) temperatures. These data confirm the
differences in wt-like p53 expression in RPMI 8226 and HS Sultan cells
cultured at permissive rather than restrictive temperatures.
Effect of CD40 activation on p53 transactivation in RPMI 8226 and HS Sultan MM cell lines After demonstrating the tsp53 mutations in RPMI 8226 and HS Sultan cell lines and the differences in wtp53 protein expression at permissive rather than restrictive conditions, we used p21-luciferase reporter assays to assess directly the temperature-dependent specific p53 transactivation function. RPMI 8226 and HS Sultan cells were transfected with either PG13-luc, plasmid that contains 13 copies of a normal p53-binding site, or with MG15-luc, plasmid that contains 15 copies of a mutated p53-binding site. Luciferase assays were performed in cells cultured in media or with NIH3T3/CD40LT for up to 40 hours at restrictive and permissive temperatures. Baseline (time 0 hour) normalized luciferase activity was increased in RPMI 8226 (Figure 2A) and HS Sultan (Figure 2B) cells transfected with PG13-luc, but not with MG15-luc, at permissive rather than restrictive temperatures. In cultures with NIH3T3/CD40LT at permissive (but not restrictive) temperatures, marked increases in normalized luciferase activity were observed in PG13-luc-transfected RPMI 8226 at 40 hours (Figure 2A) and HS Sultan cells at 16 hours (Figure 2B), but not in cells transfected with MG15-luc. These data provide strong evidence that CD40 activation induces functional wtp53 in a temperature-dependent fashion in these tumor cells.
Induction of p53 and p21 transcription and expression by CD40 activation of RPMI 8226 and HS Sultan MM cell lines To assess the biologic significance of wtp53 activation induced by CD40 activation in RPMI 8226 and HS Sultan cells at permissive temperatures, we assayed for the CD40L-triggered induction of p53 and p21 mRNA with RPA and protein expression using Western blot analysis. Probing with -actin probe and DM 1A antitubulin mAb served as a
control for RPA and Western blot analysis, respectively. The relative
intensity of expression was assessed using imaging densitometry and was
normalized against the expression in control cells cultured in media
without NIH3T3/CD40LT. Relevant data of CD40-triggered tumor cells are
presented as normalized mRNA or protein expression in Figure
3. CD40L triggered increased p53 and p21
mRNA (Figures 3A, 3C) and protein (Figures 3B, 3D) expression in both
MM cell lines when cultured at permissive, but not at restrictive,
temperatures. Specifically, in cultures at 30°C, CD40 activation
triggered 1.94- and 2.07-fold increases in p53 mRNA in RPMI 8226 (Figure 3A) and HS Sultan (Figure 3C) cells. CD40 activation also
induced 2.96- and 2.87-fold increments in p21 mRNA in RPMI 8226 (Figure
3A) and HS Sultan (Figure 3C) cells, respectively, in cultures at
30°C but not at 37°C. Similar trends were noted in p21 and p53
protein expression. Specifically, after culturing with CD40L, p53
protein expression was increased 1.68- and 3.53-fold in RPMI 8226 (Figure 3B) and HS Sultan (Figure 3D) cells. Similarly, p21 protein
expression was increased 2.26- and 2.09-fold in RPMI 8226 (Figure 3B)
and HS Sultan (Figure 3D) cells, respectively, by CD40 activation at
30°C, but not at 37°C.
Effect of temperature on viability of CD40-activated RPMI 8226 and HS Sultan MM cell lines Because RPMI 8226 and HS Sultan cells underwent tsp53 mutations with temperature-dependent differences in p53 transactivation and p53 and p21 mRNA and protein expression, we studied the effect of temperature on the CD40 activation of these tumor cells. First we examined viable cell density, assessed by trypan blue exclusion. As seen in Figure 4A, the density of viable CD40-activated RPMI 8226 cells cultured at 37°C increased as much as 11-12 × 106 cells/mL at 144 hours. Tumor cells cultured in media, with NIH3T3/wt, or with NIH3T3/vt showed maximal growth densities between 5-6 × 106 cells/mL at 96 hours to 144 hours. In contrast, CD40-activated RPMI 8226 cells cultured at 30°C exhibited only modest growth to 1-1.5 × 106 cells/mL at 48 hours, with the number of viable cells decreasing to below baseline at 144 hours. Tumor cells cultured in media, with NIH3T3/wt, or with NIH3T3/vt showed slow but sustained growth to approximately 1-1.5 × 106 cells/mL continuing to 144 hours (Figure 4B). Similar growth patterns were observed in HS Sultan cells. Specifically, the density of viable CD40-activated HS Sultan cells cultured at 37°C increased rapidly to 3-4 × 106 cells/mL at 48 hours. Cells cultured in media, with NIH3T3/wt, or with NIH3T3/vt demonstrated slower growth, with a maximum viable cell density of 3 × 106 cells/mL at 96 hours to 144 hours (Figure 4C). As seen in Figure 4D, growth of CD40-activated HS Sultan cells cultured at 30°C was reduced to 0.5-1.0 × 106 cells/mL at 144 hours, whereas cells cultured in media, with NIH3T3/wt, or with NIH3T3/vt demonstrated sustained growth to approximately 1.0-1.5 × 106 cells/mL at 144 hours. These data confirm that CD40 activation results in differences in viable cell numbers of RPMI 8226 and HS Sultan cells, which are temperature dependent.
Effect of temperature on proliferation of CD40-activated RPMI 8226 and HS Sultan MM cell lines To confirm the effect of temperature on CD40-induced proliferation of RPMI 8226 and HS Sultan cells, we performed 3H-TdR incorporation assays on tumor cells cultured at restrictive temperatures (37°C) and at permissive temperatures (30°C). Sustained increases in DNA synthesis were observed in CD40-activated RPMI 8226 cells cultured at 37°C, whereas tumor cells cultured in media, with NIH3T3/wt, or with NIH3T3/vt showed maximal 3H-TdR incorporation at 96 hours to 144 hours (Figure 5A). In contrast, proliferation of CD40-activated RPMI 8226 cells cultured at 30°C peaked at 48 hours and decreased to 144 hours, whereas tumor cells cultured in media, with NIH3T3/wt, or with NIH3T3/vt demonstrated slow, sustained growth to 144 hours (Figure 5B). Similar patterns of DNA synthesis were observed in HS Sultan cells. For example, maximum proliferation of CD40-activated HS Sultan cells cultured at 37°C was observed at 48 hours to 72 hours (Figure 5C). Tumor cells cultured in media, with NIH3T3/wt, or with NIH3T3/vt showed slow, sustained increases in 3H-TdR incorporation, which peaked at 96 hours to 144 hours. In contrast, DNA synthesis of CD40-activated HS Sultan cells cultured at 30°C peaked at 48 hours and decreased to 144 hours, whereas these cells cultured in media, with NIH3T3/wt, or with NIH3T3/vt demonstrated sustained increases in DNA synthesis up to 144 hours (Figure 5D). These data further support the functional significance of the observed tsp53 in RPMI 8226 and HS Sultan cells.
Effect of temperature on cell cycle profile of CD40-activated RPMI 8226 and HS Sultan MM cell lines Because cell proliferation was induced by CD40 activation in RPMI 8226 and HS Sultan cells cultured at restrictive temperatures (37°C) but growth arrest was observed in these tumor cell lines cultured at permissive temperatures (30°C), we next studied the effect of temperature on the cell cycle profile, assessed using PI staining, after CD40 activation of each MM cell line. As can be seen in Figure 6A, the profile of RPMI 8226 cells cultured at 37°C for 24 hours in media, with NIH3T3/wt, or with NIH3T3/vt was similar, whereas RPMI 8226 cells cultured with NIH3T3/CD40LT demonstrated G1/S transition. In contrast, CD40-activated RPMI 8226 cells cultured at permissive temperatures (30°C) for 144 hours showed increased subG1 phase cells compared with cells cultured in media, with NIH3T3/wt, or with NIH3T3/vt.
In this study we report that CD40 activation of human MM-derived cell lines can induce either proliferation or growth arrest, depending on their p53 status. We characterized 2 MM cell lines that had naturally occurring tsp53 mutations, and we used p21-luciferase reporter assays in these 2 cell lines to demonstrate that CD40 activation increased p53 transactivation function at permissive, but not at restrictive, temperatures. Moreover, p53 and p21 mRNA expression and protein expression was increased by CD40 activation of these MM cells cultured at permissive, but not at restrictive, temperatures. Of importance, we demonstrated that CD40 activation of these tumor cells cultured at restrictive temperatures was associated with increased numbers of viable tumor cells, tumor cell proliferation, and G1/S transition but that CD40 activation of MM cells cultured at permissive temperatures was associated with growth arrest and increased subG1 phase cells. These data confirmed that CD40 activation mediated cell cycle regulation in a p53-dependent mechanism.
Submitted June 9, 1999; accepted September 28, 1999.
Supported by the National Institutes of Health grants CA50947 and CA78378; the Kraft Family Research Fund; the Health Manpower Development Plan Fellowship, Ministry of Health; the National Medical Research Council-Shaw Foundation Medical Research Fellowship; the National Medical Research Council-Singapore Totalisator Board Medical Research Fellowship; and the Singapore General Hospital Medical Research Fellowship, Singapore.
Reprints: Kenneth C. Anderson, Department of Adult Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; e-mail: kenneth_anderson{at}dfci.harvard.edu.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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  Y. Nefedova, D. M. Sullivan, S. C. Bolick, W. S. Dalton, and D. I. Gabrilovich Inhibition of Notch signaling induces apoptosis of myeloma cells and enhances sensitivity to chemotherapy Blood, February 15, 2008; 111(4): 2220 - 2229. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) and with
formalin-fixed NIH3T3/CD40LT (
), NIH3T3/wt (
), or NIH3T3/vt (
)
for up to 144 hours. Tumor cells were cultured at 37°C (A, C) or
30°C (B, D), and viable cell density was assessed at 0, 2, 8, 16, 24, 36, 48, 72, 96, 120, and 144 hours using trypan blue exclusion.
Experiments were performed in triplicate, and results are expressed as
mean ± SEM.
