Reprogramming of a melanoma genome by nuclear transplantation

Abstract

Accumulating evidence shows that tumor formation is accompanied by both epigenetic and genetic alterations of the genome (Hahn and Weinberg 2002; Jones and Baylin 2002; Felsher 2003; Egger et al. 2004). Unlike genetic changes, epigenetic changes do not alter the primary DNA sequence and are therefore reversible. Examples of epigenetic modifications are the methylation of DNA and histones, the acetylation/deacetylation of histones, and the packing of chromatin into euchromatic and heterochromatic regions (Li 2002). Epigenetic modifications play an important role during normal development by regulating gene expression through stable activation or silencing of differentiation-associated genes. Similarly, epigenetic changes can promote cell proliferation, inhibit apoptosis, and induce angiogenesis during tumorigenesis by activating oncogenes and silencing tumor suppressor genes (Felsher 2003). For example, the p16 and VHL tumor suppressor genes are frequently silenced in human cancer by methylation of their promoter regions (Jones and Baylin 2002). Moreover, the treatment of tumor cells with methylation- and histone-modifying drugs can inhibit malignancy and this inhibition correlates with the reactivation of important tumor suppressor loci (Jones and Baylin 2002; Egger et al. 2004).

Epigenetic and genetic changes often act in concert during neoplasia. For example, potent oncogenes such as MYC, FOS, and PML-RAR can directly or indirectly interact with proteins that regulate epigenetic modifications such as DNA methyltransferases, histone methyltransferases, and histone acetylases/deacetylases to modulate gene expression (Bakin and Curran 1999; Di Croce et al. 2002; Jones and Baylin 2002; Ogawa et al. 2002; Felsher 2003; Frank et al. 2003). In agreement, animal models deficient for individual components of the epigenetic machinery are more prone to genome instability and cancer. For instance, mice that harbor a hypomorphic allele of the Dnmt1 methyltransferase gene consistently succumb to thymomas, possibly through the induction of chromosomal abnormalities (Gaudet et al. 2003). Likewise, the lack of the histone methyltransferase Suv39h has been associated with an increased tumor risk and genomic instability (Peters et al. 2001). These observations strongly suggest that epigenetic and genetic alterations together contribute to the neoplastic state.

Tumors develop in the context of a particular developmental state, and thus, epigenetics may also influence tumorigenesis through its effects on differentiation. Consistent with this notion, differentiation-inducing drugs can cause regression of some tumors such as all-trans retinoic acid in acute promyelocytic leukemia (Sanz et al. 1998). Moreover, many bona fide oncogenes are tumorigenic only in specific cell lineages, suggesting the requirement for a tissue-specific epigenetic environment that is permissive for an oncogene's tumorigenic potential (Felsher 2003). For example, in a MYC-inducible osteosarcoma mouse model, it has been demonstrated that expression of the MYC oncogene causes tumors in immature osteoblasts, but induces apoptosis in differentiated osteocytes (Jain et al. 2002), an observation that further supports the idea that the differentiation state and thus epigenetic conformation of a tumor cell may determine whether a cell manifests a malignant phenotype or not.

Nuclear transplantation (NT) can reprogram a terminally differentiated cell into a pluripotent embryonic cell that can direct development of an organism (Wilmut et al. 1997; Wakayama et al. 1998). This is accomplished by resetting the epigenetic modifications associated with differentiation to a state equivalent to that of a zygote (Hochedlinger and Jaenisch 2002b), while genetic changes remain unaltered (Hochedlinger and Jaenisch 2002a). Thus, NT provides a tool to selectively reprogram the epigenetic state of a cellular genome without altering its genetic constitution in order to globally analyze the impact of epigenetics on tumorigenesis. Historic experiments in frogs have demonstrated that kidney carcinoma nuclei can be reprogrammed to support early development to the tadpole stage (McKinnell et al. 1969). A similar result was recently obtained in mice where nuclei from a medulloblastoma cell line were able to direct early development, albeit with low efficiency, resulting in arrested embryos (Li et al. 2003). However, these experiments did not unequivocally demonstrate that the clones were derived from cancer cells as opposed to contaminating nontransformed cells (Carlson et al. 1994). Moreover, the experimental setup did not allow the distinction between abnormalities caused by the nuclear transfer procedure versus abnormalities caused by the donor nucleus.

Here, we have taken an alternate approach to investigate whether the reprogramming activity of the oocyte can reverse the cancer phenotype of a tumor genome and establish developmental pluripotency (Fig. 1). Following nuclear transfer of different tumor cells, clones were allowed to develop to blastocysts and then explanted in tissue culture to derive embryonic stem (ES) cells. The resulting ES cells (hereafter denoted as NT ES cells) were then analyzed to confirm the tumor cell origin and tested in multiple assays for their developmental and tumorigenic potential. This modified cloning procedure (1) circumvents abnormalities associated with nuclear transfer (Hochedlinger and Jaenisch 2003) and (2) permits a detailed analysis of the developmental (Hochedlinger and Jaenisch 2002a; Rideout et al. 2002; Eggan et al. 2004) and tumorigenic potential of the reprogrammed nucleus.

Figure 1.

Two-step cloning procedure to produce mice from cancer cells. Different tumor cells were used as donors for nuclear transfer into enucleated oocytes. Resultant blastocysts were explanted in culture to produce ES cell lines. The tumorigenic and differentiation potential of these ES cells was assayed in vitro by inducing teratomas in SCID mice (1), and in vivo by injecting cells into diploid (2) or tetraploid (3) blastocysts to generate chimeras and entirely ES-cell-derived mice, respectively.

Tumors were classified by H&E staining of histological sections and immunohistochemical analysis for melanoma specific markers S-100 and TRP-1, the rhabdomyosarcoma-specific marker desmin, or MPNST markers GFAP and S-100. Responsiveness of tumors to doxycyline was assessed either by regression of primary tumors upon doxycycline withdrawal or by doxycycline dependent growth of established tumor cell lines after transplantation into SCID mice. Expression of the RAS transgene was determined by RT-PCR that specifically detects the transgene but not the endogeneous RAS locus (data not shown). Boxes shaded in dark gray mark tumors of nonmelanocyte origin. (N/A) Not analyzed; (MPNST) Malignant peripheral nerve sheath tumor.

Acknowledgments

Notes

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1213504.

Corresponding authors.