Original ArticlesNormal and Rearranged PAX3 Expression in Human Rhabdomyosarcoma
Introduction
PAX genes contain a paired box motif that encodes a 128 amino acid DNA binding domain [1]. In addition to the paired-region, some genes of the PAX family, including PAX3, contain one or two other highly conserved sequences: the octapeptide motif and the homebox. PAX3 maps to chromosome 2q35 and shares a high degree of sequence homology with PAX7 2, 3.
The nuclear localization, the presence of a specific DNA binding motif, and their expression during embryogenesis suggest that PAX proteins may act as transcriptional modulators regulating embryonal development 4, 5.
During mouse embryogenesis the PAX3 gene is transiently expressed in the neural tube, within somites, and in limb bud mesenchyme. In muscle progenitor cells PAX3 is expressed before myogenic transcription factors, such as MyoD, Myogenin, and Myf5 and gradually declines during muscle differentiation [6]. Recently, the expression of some PAX genes was described in murine and human adult tissues, suggesting that PAX genes may also have an important role in differentiation maintenance 7, 8. Moreover, oncogenic potential of some murine PAX proteins, including PAX3, has been demonstrated in vitro and in vivo [9].
Human PAX3 is rearranged in the specific t(2;13) (q35;q14), characteristic of alveolar rhabdomyosarcoma (ARMS) [10]. The rearrangement breakpoint occurs downstream of the three conserved domains: the paired domain, the octapeptide, and the homeodomain. It results in a fusion between PAX3 and FKHR genes, the latter being a member of the family of the fork-head transcription factors, mapped to 13q14 11, 12. As a consequence of the rearrangement two chimeric transcripts originate: the 5′ region of PAX3, containing the DNA binding domain, is fused in frame with the 3′ sequence of FKHR in the derivative 13, der(13), whereas the 3′ region of PAX3 is fused with the 5′ region of FKHR in der(2). The 97-kD fusion protein derived from the der(13) transcript has been well characterized. It consists of the intact DNA binding domain of the PAX3 and of the distal half of the fork-head domain plus the C-terminal region of FKHR. It is located in the nucleus, and it is believed to recognize PAX3 target sequences 2, 13. The fusion protein shows a more potent transcriptional activity than the wild type PAX3, in vitro [13]. Similarly to PAX3, the PAX3-FKHR fusion protein seems to possess dose-dependent transcriptional modulation activity [14]. These characteristics led to the hypothesis that the PAX-3-FKHR chimeric product might play a role in the pathogenesis of ARMS and that it might interfere with the functions of the wild type PAX3 13, 14, 15.
Although some authors have studied the chimeric transcript der(13) derived from the t(2;13)(q35;q14) in human RMS by reverse transcriptase polymerase chain reaction (RT-PCR) 16, 17, 18and fluorescence in situ hybridization (FISH) analysis [19], no data are available on wild type PAX3 and der(2) transcript expression, other than in the RMS cell lines 11, 12, 20.
We studied by RT-PCR the prevalence of the t(2;13) and the sensitivity of both chimeric transcripts as markers of this translocation in a series of pediatric RMS. Furthermore, we evaluated whether the presence of the wild type PAX3 transcript showed any relationship with the histologic subtype or PAX3-FKHR expression.
Section snippets
Cell Lines
The ARMS cell line RH30, carrying the t(2;13)(q35;q14), and the embryonal RMS (ERMS) cell line RD, which does not carry any specific chromosomal translocation, were purchased from ATCC (Rockville, MD). Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (Seromed, Berlin, Germany), 100 IUI/ml Penicillin G and 100 μg/ml Streptomycin. All cell lines were mycoplasma free.
Tumor Samples
Specimens were obtained from the Italian Association of Pediatric Hematology/Oncology Soft Tissue Tumor Bank at
Results
We studied the expression of the wild type PAX3 and the chimeric products originating from the der2 and der13 in 23 RMS tumor samples and 1 leiomyosarcoma obtained from the Italian Soft Tissue Tumor Bank. Fifteen RMS were alveolar, 7 embryonal, and 1 was a spindle cell RMS. In addition, we studied normal human fetal and adult skeletal muscles. The cell lines RH30 and RD were used as a positive and a negative control for the fusion transcripts, respectively.
The wild type FKHR transcript was
Discussion
We studied by RT-PCR the pattern of expression of the wild type PAX3 and of the chimeric products derived from the t(2;13) reciprocal translocation, in a series of RMS of childhood.
The chimeric transcripts originating from the t(2;13) (q35;q14) or from the rare variant t(1;13)(p36;q14) were found only in ARMS, confirming the specific association of the t(2;13) and t(1;13) with that histologic subtype. Among the 15 ARMS we detected at least one of the chimeric transcripts originating from
Acknowledgements
E. Frascella is supported by a fellowship from Associazione Italiana per la Ricerca sul Cancro (AIRC).
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