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Distinct Expression Patterns of the p53-Homologue p73 in Malignant and Normal Hematopoiesis Assessed by a Novel Real-Time Reverse Transcription-Polymerase Chain Reaction Assay and Protein Analysis¹

Department of Hematology/Oncology, Charité/Virchow-Clinic, Humboldt-University, 13353 Berlin, Germany [U. R. P., K. A. K., G. B., U. L, C. A. S.], and Department of Clinical Research, University and Inselspital Berne, 3010 Berne, Switzerland [M. P. T., A. T., M. F. F.]

	ABSTRACT

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The role of the recently identified first p53-homologue, p73, in neoplastic transformation is unknown. To elucidate p73 gene expression in hematopoiesis, we investigated samples from chronic myeloid leukemia (CML) and acute myeloid leukemia patients, leukemia cell lines, as well as mature and immature normal hematopoietic cells by real-time quantitative RT-PCR and Western blot analysis. We found a distinct p73 expression profile with highest p73 mRNA transcript levels in hematopoietic malignancies such as CML blast crisis and acute myelogenous leukemia versus CML chronic phase and normal controls. Mono- and biallelic p73 expression was found in both normal and malignant hematopoiesis. p73 protein was expressed at various levels in leukemia samples and cell lines but could not be detected in any normal controls tested. Our results point to a distinct yet undefined role of p73 in the pathogenesis of myeloid neoplasms.

	Introduction

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The p53 tumor suppressor gene plays a pivotal role in preventing uncontrolled growth and neoplastic transformation in cells by modulating critical cellular processes such as cell cycle arrest and apoptosis. In more than 50% of human cancers, normal p53 function is abrogated by gene mutations, deletions, or interactions with viral or cellular oncoproteins (1) . p53 alterations are important determinants of treatment efficacy and outcome in cancer, including various hematological neoplasms (2) .

CML³ is a clonal myeloproliferative disorder associated with the t(9;22)(q34;11) translocation generating the BCR-ABL fusion gene. AML is mostly caused by a variety of clonal chromosomal aberrations that either increase the proliferative capacity of the cells or keep the cells arrested in an immature proliferative state. Alterations of the p53 gene play a role during CML disease progression (3) and, albeit to a lower extent, may contribute to AML development (4) .

Recently, the first p53-related gene, p73, was identified on chromosome 1p36. It consists of 14 exons encoding a 2.2-kb mRNA and a M_r 73,000 protein and may be spliced into several isoforms, which are expressed in various tissues (5 , 6) . In addition, p73 seems to be predominantly expressed from the maternal allele, whereas the paternal allele remains silent, most probably due to genetic imprinting (5 , 7) . p73 is thought to be a candidate tumor suppressor based on: (a) the genomic localization in a region frequently altered in neuroblastoma and other human cancers (5) ; (b) its significant structural homology to core domains of the p53 protein; and (c) transcriptional functions, e.g., p21^Waf1-induced growth arrest and apoptosis, which are remarkably similar to p53 (5 , 8 , 9) . Unlike p53, p73 does not appear to be induced by DNA damage (5) , and it shows disparate interactions with viral oncoproteins (10 , 11) .

Recent data on p73 expression suggest a possible role of the gene in the pathogenesis of several solid tumors including neuroblastoma, lung cancer, prostate cancer, and renal cell carcinoma (5 , 7 , 12 , 13) . However, data on p73 expression in normal and malignant hematopoietic cells are scarce (14, 15, 16) . We investigated the expression of the p73 gene in normal and malignant hematological tissue, including a panel of leukemia cell lines, as well as fresh samples from CML and AML patients. Healthy donor peripheral blood leukocytes, granulocytes, and lymphocytes, as well as CD34⁺ hematopoietic progenitor cells, served as normal controls. We assessed p73 mRNA transcript levels using a novel real-time quantitative RT-PCR and p73 protein expression by Western blot analysis.

	Materials and Methods

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Samples and Cell Lines.
Our study comprises samples from patients with CML-CP (n = 22), CML-AP/BC (n = 9), CML in complete (molecular) remission after allogeneic bone marrow transplantation (CML-BMT, n = 12) with a median interval from BMT to sample collection of 136 days (range, 43–2356 days), from AML (n = 41) as well as from healthy donor peripheral blood leukocyte (n = 9), granulocyte (n = 8), lymphocyte (n = 5), and CD34⁺ progenitor cell (n = 4) samples. All samples were obtained using standard collection and separation procedures (17 , 18) . We also included seven myeloid (HL 60, U 937, K 562, KG 1, EM 3, THP 1, and NB 4) and six lymphoid (CEM, Jurkat, Molt 4, Raji, Daudi, and Nalm 6) leukemia cell lines obtained from the American Type Culture Collection (Rockville, MD) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) in our analysis. Cell lines were grown as described previously (18) .

Real-Time Quantitative RT-PCR.
Real-time quantitative RT-PCR is based on fluorescence emission by a sequence-specific, nonextendable probe labeled with a 5'-reporter and 3'-quencher dye. During the extension phase, the probe is cleaved by the 5'-exonuclease activity of Taq DNA polymerase. Subsequent separation of quencher and reporter dye releases a fluorescence signal. Background fluorescence is calculated during the first 15 amplification cycles. TaqMan PCR becomes positive when the fluorescence signal exceeds the 10-fold SD of background fluorescence, which marks the threshold cycle (Ct). A 152-bp p73 cDNA standard was amplified from K562 cells using primers spanning exons 5 to 6 without amplification of genomic sequences (p73-715F 5'-ACTTCAACGAAGGACAGTCTGCT and p73-856R 5'-AATTCCGTCCCCACCTGTG). The amplicon was gel extracted, purified (Qiagen, Hilden, Germany), and cloned into a pCR2.1 vector (Invitrogen, Leek, the Netherlands). After digestion with HindIII and XbaI, the insert was reamplified, purified, and quantified spectrophotometrically. Molecule concentrations were calculated, and serial dilutions of standard DNA ranging from 10⁷ to 10^-2 molecules per 2-µl reaction volume were prepared. To correct for RNA quality differences, we measured ß-actin mRNA expression in parallel and calculated the ratio between the absolute number of p73 and ß-actin mRNA transcripts in a given sample (17) . Our assay allowed the reproducible detection of 10 p73 mRNA transcripts and 10 ß-actin mRNA transcripts per reaction, respectively (Fig. 1) . Copy numbers <10 were not quantified. Random-primed, first-strand cDNA was synthesized from total RNA (1 µg/20 µl reaction) using the 1st Strand cDNA Synthesis kit (Roche Diagnostics, Rotkreuz, Switzerland). The TaqMan probe (5'-CCTCATCCGCGTGGAAGGCAATAATCTC) was 5'-labeled with carboxy-fluorescein phosphoramidite as reporter dye, 3'-labeled with carboxy-tetramethyl-rhodamine for quenching, and 3'-phosphate groups prevented probe extension (TIB Molbiol, Berlin, Germany). PCR was carried out with 5 µl of 10x PCR buffer, 4.5 mM MgCl₂, 0.8 mM deoxynucleotide triphosphate, 1 µM carboxy-X-rhodamine, 0.5 µM of each primer (p73-715F and p73-856R), 1 µM probe, 1.25 units of a temperature release Taq DNA polymerase (Platinum DNA polymerase; Life Technologies, Inc., Karlsruhe, Germany), and 100 ng of sample cDNA in a total volume of 50 µl. After an initial 5-min denaturation step at 94°C, we performed 45 cycles of denaturation at 94°C for 30 s and annealing/extension at 63°C (p73) or 67°C (ß-actin) for 60 s. Amplification and data analysis were carried out using the ABI PRISM 7700 Sequence Detection System and the Sequence Detector V 1.6.3 program (PE Applied Biosystems, Foster City, CA). Most samples were measured in duplicate.

View larger version (47K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. P73 real-time quantitative RT-PCR. A, serial dilutions of the 142-bp p73 real-time RT-PCR standard visualized by agarose gel electrophoresis. B, real-time quantitative RT-PCR showing the reproducible detection of 10 p73 mRNA transcripts per reaction. NTC, no template control. MW, molecular weight marker.

p73 Protein Analysis.
Whole-cell protein extracts were prepared by sonication for 5 min in 1% NP40 buffer and quantified spectrophotometrically (Bio-Rad, Glattbrugg, Switzerland). Total protein (130 µg) was size-fractionated on a 8% SDS-polyacrylamide gel and blotted to nitrocellulose (Bio-Rad). The membrane was blocked in 2% dry milk/TBS-T for 1 h, incubated over night at 4°C with a 1:500 dilution of the p73 polyclonal antiserum (kindly provided by Dr. D. Caput, Labege, France; Ref. 5 ), and visualized using the ECL system (Amersham, Zürich, Switzerland).

Statistics.
Because of the sample size and nonnormal distribution, only nonparametric tests were applied (StatView 4.0; Abacus Concepts, Berkeley, CA). p73 mRNA expression was analyzed in seven independent groups using the KWT with a P for significance set at 0.05. For differences between particular groups, the conservative Bonferroni procedure was performed, and the P was set at 0.0063. The remaining statistical analyses were carried out using the KWT or MWU test (significance, P <0.05).

	Results and Discussion

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p73 mRNA Expression Analysis.
We investigated p73 gene expression in leukemia cell lines, samples from CML and AML patients, as well as different types of normal hematopoietic cells. Using conventional RT-PCR, we found p73 message in 30 of 31 CML patient samples (22 of 22 CML-CP; 8 of 9 CML-AP/BC), 32 of 35 AML patient samples, 6 of 7 myeloid leukemia cell lines, 3 of 6 lymphoid leukemia cell lines, 10 of 12 samples from CML patients after allografting (CML-BMT), as well as 9 of 9 healthy donor leukocyte samples, 2 of 8 granulocyte samples, 5 of 5 lymphocyte samples, and 4 of 4 CD34⁺ progenitor cell samples.

To obtain more detailed information, we performed a real-time quantitative RT-PCR assay showing a higher sensitivity than our conventional RT-PCR assay. We discovered a distinct p73 mRNA expression pattern in patient samples, leukemia cell lines, and normal hematopoietic cells (KWT, P < 0.0001) depicted as the ratio of p73 and ß-actin transcripts (Fig. 2) . In CML, we found the highest p73 mRNA expression levels in samples from CML-AP/BC patients compared with intermediate levels in CML-CP patients (MWU, P < 0.0004) and low levels in CML-BMT patients (P = 0.0003). The CML-CP group differed clearly from CML-BMT, albeit significance was not reached after the conservative Bonferroni procedure (MWU, P = 0.0075). Although some CML-CP samples expressed high p73 mRNA levels, this entity did not differ significantly from normal leukocytes and CD34⁺ hematopoietic progenitors. In three of four patients with material available before and after bone marrow transplantation, intermediate to high p73 mRNA transcript levels prior to BMT fell by the magnitude of 1 to 2 logarithmic steps after allografting (data not shown). One CML-AP/BC and one CML-BMT patient sample showed p73 mRNA copies below the quantification threshold of 10 p73 mRNA transcripts. In this respect, recent reports point to p73 promoter hypermethylation rather than genomic deletion as a possible mechanism for p73 gene silencing (14 , 16 , 19) .

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Quantitative p73 mRNA transcript analysis (median and range) in normal leukocyte and lymphocyte samples, CD34+ hematopoietic progenitor cells, CML-CP, CML-AP/BC, CML-BMT, AML, and myeloid leukemia cell lines. Malignant cells revealed overall higher p73 mRNA levels compared with normal controls. Results are depicted as the ratio of absolute p73 and ß-actin mRNA transcript numbers; bars, SD.

In normal peripheral blood leukocytes, lymphocytes, and CD34⁺ progenitor cells, p73 mRNA was expressed at relatively low levels but was still detectable by conventional RT-PCR and real-time quantitative RT-PCR. Most granulocyte samples expressed ß-actin at significantly lower levels than any other cell type (MWU, P = 0.0001) but comparable p73 transcript numbers, thus yielding relatively high p73:ß-actin ratios, which probably overestimated the actual p73 mRNA expression. We therefore excluded granulocyte samples from p73 mRNA quantification. Our data on p73 mRNA expression in hematopoietic cells correspond to previous findings in solid tumors, where p73 expression assessed by qualitative PCR appeared to be stronger in malignant tissues compared with their normal counterparts (12 , 13) .

p73 Allele-specific Expression Analysis.
The p73 gene is located in a chromosome region that seems to be regulated by genetic imprinting sustaining predominantly the maternal (GC) allele (5) . Analysis of p73 allele expression revealed that the majority of our samples with p73 mRNA detectable exclusively expressed the GC allele (74 of 102; Table 1 ). Some samples showed coexpression of GC and AT alleles (20 of 102) or, rarely, monoallelic expression of the AT allele (8 of 102). Interestingly, in more than half of the AML patients (19 of 33), the AT allele was expressed, in some cases, at considerable levels. In CML patients, we found expression of the AT allele only in cases with AP/BC (two of eight). At first sight, these data may support findings in solid tumors, where it was assumed that expression of the paternal (AT) allele might be tumor specific (5 , 7 , 12) . However, in this study, the AT alleles were also expressed at various levels in healthy donor peripheral blood leukocyte samples (four of nine), CD34⁺ progenitor cells (two of four), and lymphocytes (one of five). In addition, the allelic expression status did not influence p73 mRNA transcript levels assessed by our quantitative RT-PCR analysis. Our data show that mono- or biallelic expression is not restricted to hematopoietic neoplasms, suggesting that p73 allele status may not be relevant for leukemogenesis. However, the functional significance of p73 allele expression still awaits to be elucidated, and considerable intertissue variation might exist (14 , 15 , 20) .

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Analysis of p73 protein in hematopoietic cells including myeloid leukemia cell lines (THP-1, EM-3, K-562, KG-1, HL-60, and NB-4), lymphoid leukemia cell lines (Nalm-6, CEM, and Jurkat), AML patient samples, as well as healthy donor granulocyte and CD34+ progenitor cell samples. Equal amounts of total protein were loaded. K-562 and Jurkat cells served as positive and negative control, respectively. Myeloid leukemia cell lines and patient samples expressed p73 protein at various levels, ranging from high to undetectable. All lymphoid leukemia cell lines and normal controls tested did not express p73 protein.

In conclusion, we found a wide range of p73 mRNA transcript levels in hematopoietic neoplasms with pronounced expression, particularly in advanced stages of CML, some AML patients, most myeloid, and some lymphoid leukemia cell lines. In contrast, normal hematopoietic cells showed a comparably narrow range with lower expression of p73 message. However, we noted a partial overlap of p73 transcript levels between AML and CD34⁺ progenitor cell samples. Our protein data confirmed the results of RNA quantitation. Given the fact that p73 mRNA expression parallels p73 protein expression and mutations of the p73 gene are rare, p73 mRNA quantification may provide at least some indirect information on the function of the gene in a given sample. We believe that the p73 gene plays a distinct yet undefined role in leukemogenesis, particularly in myeloid leukemias. Additional studies, in particular gene knock-out experiments, may clarify the position of p73 within the oncogene and tumor suppressor gene network.

	ACKNOWLEDGMENTS

 
We thank Jutta Laser, Bärbel Pawlaczyk-Peter, and Madeleine Oestreicher for excellent technical assistance and Dr. Daniel Caput for provision of the p73 antiserum.

	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 This work was supported by Grant KI 9605 from the Bundesministerium Für Bildung und Forschung, a grant from the José Carreras Foundation, Grant 31-43458.95 from the Swiss National Foundation, and Grant KFS 156-9-1995 from the Swiss Cancer League.

² To whom requests for reprints should be addressed, at Department of Hematology/Oncology, Charité/Virchow-Clinic, Humboldt-University, Augustenburger Platz 1, D-13353 Berlin, Germany. Phone: 49-30-450-53663; Fax: 49-30-450-53925; E-mail: christian.schmidt{at}charite.de

³ The abbreviations used are: CML, chronic myeloid leukemia; CP, chronic phase; AP, acceleration phase; AML, acute myeloid leukemia; BC, blast crisis; BMT, bone marrow transplantation; KWT, Kruskal-Wallis test; MWU, Mann-Whitney U test.

Received 6/ 7/99. Accepted 7/19/99.

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