Normal and Rearranged PAX3 Expression in Human Rhabdomyosarcoma

Abstract

PAX3, a member of the PAX-gene family, encodes a nuclear transcription factor that is transiently expressed in the neural tube and in muscle progenitor cells and regulates embryonal development in the mouse. Together with the FKHR gene it is involved in the t(2;13)(q35;q14), a specific translocation associated with alveolar rhabdomyosarcoma (ARMS). As a consequence of the rearrangement two chimeric transcripts originate: FKHR-PAX3 and PAX3-FKHR. We studied the expression of wild type PAX3 and the chimeric transcripts originating from the t(2;13) in a series of 23 rhabdomyosarcomas (RMS) of childhood, by reverse transcriptase polymerase chain reaction (RT-PCR). Wild type PAX3 was detected in 48% of the RMS, whereas another 39% were positive only after nested PCR. Normal adult skeletal muscle showed a very weak expression of PAX3, but fetal muscle did not express PAX3. PAX3-FKHR was found in 11 of 15 alveolar RMS, 7 of which were positive also for the reciprocal transcript, whereas no RMS expressed FKHR-PAX3 alone. These results confirm that the PAX3-FKHR transcript is specifically associated with the alveolar RMS and that it is a more sensitive marker of the t(2;13) than the reciprocal product FKHR-PAX3. Furthermore, the finding of PAX3 expression with or without PAX3-FKHR transcript in the great majority of the cases raises the question of whether PAX3 expression could play a role in the pathogenesis of RMS.

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.