Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: Pediatr Res. 2012 Jan;71(1):32–38. doi: 10.1038/pr.2011.6

A Set of Imprinted Genes Required for Normal Body Growth Also Promotes Growth of Rhabdomyosarcoma Cells

Geoffrey Rezvani 1,*, Julian CK Lui 1, Kevin M Barnes 1, Jeffrey Baron 1
PMCID: PMC3420822  NIHMSID: NIHMS396479  PMID: 22289848

Abstract

In many normal tissues, proliferation rates decline postnatally causing somatic growth to slow. Previous evidence suggests that this decline is due in part to declining expression of growth-promoting imprinted genes including Mest, Plagl1, Peg3, Dlk1, and Igf2. Embryonal cancers are composed of cells that maintain embryonic characteristics and proliferate rapidly in childhood. We hypothesized that the abnormal persistent rapid proliferation in embryonal cancers occurs in part because of abnormal persistent high expression of growth-promoting imprinted genes. Analysis of microarray data showed elevated expression of MEST, PLAGL1, PEG3, DLK1, and IGF2 in various embryonal cancers, especially rhabdomyosarcoma, compared to non-embryonal cancers and normal tissues. Similarly, mRNA expression, assessed by real-time PCR, of MEST, PEG3, and IGF2 in rhabdomyosarcoma cell lines was increased compared to non-embryonal cancer cell lines. Furthermore, siRNA-mediated knockdown of MEST, PLAGL1, PEG3, and IGF2 expression inhibited proliferation in Rh30 rhabdomyosarcoma cells. These finding suggest that the normal postnatal downregulation of growth-promoting imprinted genes fails to occur in some embryonal cancers, particularly rhabdomyosarcoma, and contributes to the persistent rapid proliferation of rhabdomyosarcoma cells, and, more generally, that failure of the mechanisms responsible for normal somatic growth deceleration can promote tumorigenesis.

Introduction

In mammals, somatic growth is rapid in embryonic and early postnatal life but decelerates with age. This decline in growth rate is due, in large part, to a decrease in the rate of cell proliferation(1–4). We recently showed evidence that the decline in proliferation is driven by an extensive genetic program that occurs coordinately in multiple tissues during early postnatal life in mice (5, 6). This complex program includes the downregulation of multiple growth-promoting imprinted genes, including Igf2, Plagl1, Mest, Peg3, Dlk1, Gtl2, and Slc38a4 (7). Targeted ablation of these imprinted genes in mice causes decreased body size at birth, indicating that they are required for rapid embryonic growth (8–11). However, their expression subsequently declines markedly, probably contributing to normal postnatal growth deceleration (5, 6).

Although Mest, Peg3, Plagl1, and Igf2 are all required for normal somatic growth in mice, the molecular functions of the genes products are quite disparate. Igf2 encodes insulin-like growth factor-II, a secreted protein that interacts with the type I insulin-like growth factor receptor to promote growth in a wide variety of cell types. Knockout of Igf2 causes mice to be small in length and weight (8). Plagl1, pleiomorphic adenoma gene-like 1, encodes a C2H2 zinc-finger transcription factor. Unlike related genes, Plag1 and Plagl2, which act as proto-oncogenes, Plagl1 is considered a candidate tumor suppressor because reduced expression has been observed in tumor cells and because overexpression in tumor cells can induce apoptosis and cell-cycle arrest (12). However, knockout of Plagl1 in mice causes intrauterine growth restriction as well as postnatal pulmonary dysfunction (13). Mest has sequence similarity to α/β fold hydrolases. Knockout of Mest in mice causes growth retardation of embryonic and extra-embryonic structures and abnormal maternal behavior (9). Peg3 encodes a protein with 12 Kruppel type zinc finger domains and two proline-rich periodic repeat domains. There is evidence that Peg3 promotes apoptosis and has a variety of molecular actions, including effects on Bax/p53, TNF-α, and Wnt-related pathways (14). Mice deficient in Peg3 are viable but smaller than wild-type animals, and female mutants show impaired maternal behavior including abnormal nest-building and gathering of pups (10). MEST, PEG3, PLAGL1, and IGF2 all exhibit imprinting with silencing of the maternal allele and expression of the paternal allele. It has previously been observed that a disproportionate number of imprinted genes are involved in growth regulation and particularly that paternally expressed genes tend to promote body growth (15).

Embryonal cancers are composed of cells that maintain embryonic characteristics and show abnormally rapid proliferation and other malignant features. These tumors typically present in childhood and include rhabdomyosarcoma, Wilms’ tumor, neuroblastoma, and retinoblastoma, which resemble embryonic muscle, renal, neural crest, and retinal tissue respectively. Embryonal cancers are among the most common solid extra-cranial tumors of childhood, accounting for 8–10% of childhood malignancies (16).

We hypothesized that failure of the genetic program responsible for normal growth deceleration may contribute to the rapid proliferation and immature characteristics observed in embryonal cancers. Because this program appears to suppress the rapid cell proliferation that normally occurs in embryonic tissues, we reasoned that failure of the program in a clone of cells would allow persistent rapid proliferation in postnatal life, thus contributing to tumorigenesis. As a first test of this hypothesis, we focused on the growth-promoting imprinted genes that are normally downregulated postnatally, asking whether these genes show persistently elevated expression in embryonal cancers. Then, to determine whether these genes are required for the rapid growth of rhabdomyosarcoma, we knocked down expression of these genes in cultured rhabdomyosarcoma cells and examined the effect on proliferation rate.

Methods

Microarray

We analyzed an expression microarray database from the laboratory of Dr. Javed Khan (17), which is based on 163 cancer samples (75 xenograft, 70 primary tumors, and 18 cell lines), including embryonal and non-embryonal pediatric cancers. The embryonal tumor samples include rhabdomyosarcoma (n=34), neuroblastoma (n=62), Wilms’ tumor (n=8), medulloblastoma (n=6), ependymoma (n=4). Cancers of non-embryonal origin include acute lymphoblastic leukemia (n=10), Ewing’s sarcoma (n=31), and osteosarcoma (n=8). Of the 163 samples analyzed in the microarray, 18 were cell lines (12 neuroblastoma, 3 rhabdomyosarcoma, 3 Ewing’s sarcoma), 70 were primary tumor samples (30 neuroblastoma, 21 rhabdomyosarcoma, 19 Ewing’s sarcoma) and the remainder were xenografts. Some of the cell lines, xenografts, and primary tumor samples might have been derived from a common origin. Normal pediatric tissue samples (n=19) include gastrointestinal tissue, kidneys, ureters, muscle, uterus, testes, lung, heart, brain and adrenal glands. Expression of mRNA was normalized to a combined sample of total RNA from 7 human cancer cell lines (CHP212, RD, HeLa, A204, K562, RD-ES, and CA46). For this prior study, all samples required appropriate Institutional Review Board and Material Transfer Agreement approval from the donating institution(17).

Cell culture

Rh30, Rh4, Rh28, CTR, RD-ATCC, Rh1, Daudi, K562, MEG-1, and HCT116 cell lines were cultured in RPMI 1640 (Invitrogen), and HeLa, BT474, SKBR3, TC-71, RD-ES, and U-2 OS cell lines were cultured in DMEM (Invitrogen). Both culture media were supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin (Invitrogen), 100 U/ml streptomycin (Invitrogen), and 300 μg/ml L-Glutamine (Invitrogen). Cells were cultured at 37 C in 5% CO2 and split every 3–4 days (at approximately 75% confluence for adherent cell lines) using 0.25% trypsin with 0.038% EDTA (Invitrogen) for 1 minute. Cell lines were provided by Dr. Javed Khan, NCI, NIH (Duadi, K562, MEG-1, HCT116, HeLa, BT474, SKBR3, TC-71, RD-ES, U-2 OS) and Dr. Lee Helman, NCI, NIH (Rh30, Rh4, Rh28, CTR, RD-ATCC, Rh1). Rh30 (18–20), Rh4 (20), Rh28 (20), CTR (20), and RD-ATCC (20–22) are rhabdomyosarcoma cell lines, while the remaining 13 are derived from cancers of non-embryonal origin: HeLa, cervical cancer (23); HCT116, colon (24); BT474 (25, 26) and SKBR3 (26), breast; TC-71 (27), RD-ES (28), and Rh1 (18, 29), Ewing’s sarcoma; U-2 OS, osteosarcoma (30–32); Daudi (33), Burkitt’s lymphoma; and K562 (34) and MEG-1 (35), chronic myeloid leukemia.

Quantitative real-time RT-PCR

Total RNA was isolated from cultured cells using the RNeasy kit (Qiagen, Valencia, CA), and then reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen), both according to the manufacturers’ instructions. Quantitative real-time PCR was performed using the following assays containing primers and specific intron-spanning FAM-labeled TaqMan probes (Applied Biosystems, Foster City, CA): MEST, Hs083380_91; PEG3, ZIHs00377844_m1; PLAGL1, Hs00414677; IGF2, Hs00171254_m1. An assay for 18S rRNA using a VIC/TAMRA-labeled TaqMan probe (Applied Biosystems) was used for normalization. Reactions were performed in triplicate using cDNA, TaqMan universal PCR Master Mix (Applied Biosystems), and the ABI 7900HT Sequence Detection System (Applied Biosystems), according to the manufacturer’s instructions with the following thermal cycling conditions: 1 cycle at 50 C for 2 min and 95 C for 10 min, followed by 45 cycles of 95 C for 15 s and 60 C for 1 min. The quantity of each mRNA was calculated using the formula: relative expressioni = (2)CTr/(2)CTi where r represents 18S rRNA (for internal normalization), i represents the gene of interest, and CT represents the threshold cycle. For convenience, relative expression values were multiplied by 106.

siRNA transfection

One day prior to transfection, Rh30 cells were plated in 6-well plates at a density of 200,000 cells per well to reach approximately 50% confluence at the time of transfection. Cells were transfected with siRNA (Silencer siRNA, Ambion, Austin, TX) targeting MEST, PEG3, PLAGL1, and IGF2 (Table 1), using 5 uL LipofectAMINE 2000 (Invitrogen) in 2.25 mL total volume per well in 6-well plates. Final concentrations of siRNA required to achieve maximal knockdown were optimized. A scrambled sequence siRNA of similar length (Ambion, catalog #AM4635) was used as a negative control at a concentration of 25 nmol. Cells were cultured at 37 C in 5% CO2 for an additional 48 hours after transfection, then used to isolate RNA for real-time RT-PCR as described above or treated with 3H -thymidine as described below.

Table 1.

siRNA sequences used for transfection experiments

siRNA Sense sequence (5′→3′) Antisense sequence (5′→3′) Ambion ID# Concentration (nmol)
MEST #1 GGUACAAGCAGAAUCGAUCtt GAUCGAUUCUGCUUGUACCtg 106797 75
MEST #2 GGGAUUGUUGUGUAGUCAtt UUGACUACACAACAAUCCCtt 106798 100
PEG3 #1 GCACCAGUCGAGGUCUAAAtt UUUAGACCUCGACUGGUGCtt 144245 100
PEG3 #2 CGUAACUGUUCAUAAGAAUtt AUUCUUAUGAACAGUUACGtg 144246 100
PLAGL1 #1 GGCUUUUCCGUAAACAUAUtt AUAUGUUUACGGAAAAGSStc 108090 25
PLAGL1 #2 CGCGUGUUCUGUAAUCAAtt UUUGAUUACAGAACACGCGtt 115818 100
IGF2 #1 CAAUUGUGGAACCCACAUUtt AAUGUGGGUUCCACAAUUGtg 254540 50
IGF2 #2 AAUUACCUGCCCAUUCGUCtt GACGAAUGGGCAGGUAAUUtg 238102 75
Open in a new tab

Tritiated thymidine incorporation

Cell proliferation was assessed by tritiated thymidine incorporation. Forty-eight hours after transfection with siRNA, tritiated thymidine ([Methyl-3H] thymidine in aqueous solution, 25 Ci/mmole, catalog #TRK120, Amersham, NJ) was added to the culture medium at a concentration of 1 μCi/ml. Cells were incubated for an additional 4 hours at 37 C in 5% CO2, after which cells were detached with trypsin, and incorporation of tritiated thymidine was measured by liquid scintillation counting. For each experiment, 3 replicate wells were used per siRNA. The experiment was repeated a second time and results combined.

Statistical analysis

Data are presented as mean ± SEM. Specific mRNA levels in rhabdomyosarcoma compared to non-embryonal cancers, and effects of knockdown by siRNA on mRNA levels were evaluated by one-way ANOVA. Effects of siRNA on tritiated thymidine incorporation were normalized to results from the negative control scrambled siRNA and then evaluated by two-way ANOVA to account for effects of multiple experiments. Correlation of gene expression was evaluated by linear regression using SigmaStat (Systat Software Inc., San Jose, California).

Results

To determine whether imprinted genes that have been implicated in physiological growth deceleration (6) are overexpressed in embryonal cancers, we first evaluated microarray data available from an NCI database (http://home.ccr.cancer.gov/oncology/oncogenomics/) (17) which includes embryonal tumor samples, xenografts, and cell lines. Rhabdomyosarcoma samples showed elevated expression of MEST, PEG3, PLAGL1, IGF2, and DLK1 compared to the reference RNA sample derived from 7 non-embryonal human cancer cell lines and compared to non-embryonal cancers and normal tissues (including muscle and kidney) in this database (Figure 1). Wilms’ tumor samples showed elevated expression of MEST, and IGF2 (Figure 1). Neuroblastoma samples only showed elevated expression of DLK1 (Figure 1). Ependymoma samples showed elevated expression of IGF2 (Figure 1). SLC38A4 did not appear to be overexpressed consistently by any tumor type (data not shown), and GTL2 expression could not be assessed because it was not represented in the database.

Figure 1.

Figure 1

Open in a new tab

Expression of MEST (A), PEG3(B), PLAGL1 (C), IGF2 (D), and DLK1 (E) in embryonal tumors, non-embryonal tumors and normal tissues. Data are from a NCI expression microarray database. Each bar represents a single primary tumor, xenograft, or cell line or a normal tissue sample. The plotted value is the fold difference in mRNA expression of MEST, PEG3, PLAGL1, IGF2, or DLK1 normalized to a reference RNA mixture from 7 adult, non-embryonal tumor cell lines. RMS, rhabdomyosarcoma; MB, medulloblastoma; EP, ependymoma; NB, neuroblastoma; WT, Wilms’ tumor; ALL, acute lymphoblastic leukemia; OS, osteosarcoma; ES, Ewing’s sarcoma; NT, normal tissue.

Because the microarray analysis showed elevated expression of multiple growth-promoting imprinted genes in rhabdomyosarcoma tumor samples, xenografts, and cell lines, we decided to focus subsequent studies on this embryonal cancer. To confirm the microarray findings, we first analyzed mRNA expression of MEST, PEG3, PLAGL, and IGF2 in rhabdomyosarcoma cell lines (n = 5) using real-time PCR. MEST, PEG3, and IGF2 mRNA levels were in general higher in rhabdomyosarcoma cell lines than in non-embryonal cancers (n = 11) of adult and pediatric origin (Figure 2). The corresponding difference for PLAGL1 did not reach statistical significance.

Figure 2.

Figure 2

Open in a new tab

Relative mRNA expression of IGF2 (A), MEST (B), PLAGL1 (C) and PEG3 (D) in rhabdomyosarcoma cell lines and non-embryonal cancer cell lines, measured by real-time PCR. Levels of IGF2, MEST and PEG3 mRNA were significantly higher in the 5 rhabdomyosarcoma cell lines than in 11 non-embryonal cancer cell lines. PLAGL1 did not reach significance. White bars, relative mRNA expression in rhabdomyosarcoma cell lines; black bars, relative mRNA expression in non-embryonal cancer cell lines. *, P = 0.05; **, P < 0.005; ***, P < 0.001; §, NS.

To determine whether the high expression levels of these genes contributes to the rapid proliferation of rhabdomyosarcoma cells, we used siRNA interference to suppress expression of MEST, PLAGL1, PEG3, and IGF2, in the Rh30 rhabdomyosarcoma cell line. Each gene was targeted independently with two siRNAs. Transfection of all siRNAs decreased the target mRNAs by at least 50% compared to values from a negative control siRNA, as measured by real-time PCR (Figure 3). siRNA targeting of MEST, PEG3, PLAGL1, and IGF2 decreased proliferation compared to values from a negative control siRNA, as assessed by 3H-thymidine incorporation, (Figure 4). This effect was seen for each of the two siRNAs targeting each gene, suggesting that the effect on proliferation was not due to off-target effects of the siRNA on other genes.

Figure 3.

Figure 3

Open in a new tab

Effect of IGF2, MEST, PEG3, and PLAGL1 siRNA transfection on target mRNA levels in Rh30 cells. mRNA levels were measured by real-time RT-PCR 48 hours after siRNA transfection and compared to mRNA levels in cells transfected with a negative control siRNA. Two independent siRNAs were used for each gene. All siRNAs significantly reduced expression of the target gene. White bars, transfection with negative control siRNA; black bars, transfection with siRNA #1; hatched bars, transfection with siRNA #2. * P < 0.001.

Figure 4.

Figure 4

Open in a new tab

Effects of siRNA transfection on proliferation of Rh30 rhabdomyosarcoma cells. Cells transfected with siRNA targeting MEST, PEG3, PLAGL1, and IGF2 caused significant reduction in the rate of proliferation when compared to negative control siRNA, as measured by tritiated thymidine incorporation. This effect was confirmed with a second siRNA sequence for each gene target. White bars, transfection with negative control siRNA; black bars, transfection with siRNA #1; grey bars, transfection with siRNA #2. * P < 0.001.

Next, we analyzed the microarray data to determine whether MEST, PEG3, PLAGL1, and IGF2 are regulated coordinately in rhabdomyosarcomas. In rhabdomyosarcoma tumor samples, cell lines, and xenografts there was a positive correlation between IGF2 expression and expression MEST, PEG3 and PLAGL1. Thus samples that showed higher levels of IGF2 expression tended also to show higher expression levels of MEST, PEG3, and PLAGL1 (Figure 5).

Figure 5.

Figure 5

Open in a new tab

Correlation of expression of IGF2 with expression of MEST, PEG3, and PLAGL1 in rhabdomyosarcoma primary tumors, cell lines, and xenografts. Plotted values are derived from a NCI expression microarray database and represent the fold difference in expression of the mRNA compared to a reference RNA. Samples which expressed IGF2 mRNA at high levels also expressed higher levels of MEST, PEG3, and PLAGL1, suggesting that these genes are regulated coordinately in rhabdomyosarcomas. MEST: P < 0.005, r = 0.497; PEG3: P < 0.001, r = 0.617; PLAGL1 P < 0.001, r = 0.386.

Because previous evidence suggests that PLAGL1 may regulate expression of a large network of imprinted genes (13), we analyzed the effect of siRNA-mediated knockdown of PLAGL1 on the expression of MEST, PEG3, and IGF2 in Rh30 cells. PLAGL1 knockdown did not have consistent effects on mRNA levels of any of these genes (Figure 6). Similarly, knockdown of MEST, PEG3, and IGF2 had little effect on mRNA expression of any other gene studied, except for a possible reduction in PLAGL1 expression after knockdown of PEG3 (Figure 6).

Figure 6.

Figure 6

Open in a new tab

Effect of siRNA-mediated knockdown of MEST (A), PEG3 (B), PLAGL1 (C), and IGF2 (D) expression on expression of other imprinted genes of interest. Rh30 rhabdomyosarcoma cells were transfected with siRNA targeting MEST, PEG3, PLAGL1, or IGF2 and the resulting effect on mRNA levels was assessed by real-time RT-PCR. For each target gene, two different siRNA sequences were used independently. The only consistent effect observed was that PEG3 knockdown decreased PLAGL1 mRNA levels. White bars, transfection with negative control siRNA; black bars, transfection with siRNA #1; hatched bars, transfection with siRNA #2. *, P < 0.05.

Discussion

We have previously shown evidence that the normal postnatal deceleration in mammalian body growth is driven by a genetic program that involves downregulation of many growth-promoting genes (5–7). Some of these downregulated, growth-promoting genes are imprinted, including Igf2, Plagl1, Mest, Peg3, Dlk1, and Slc38a4. In the current study, our analysis of available microarray data suggested that some of these imprinted genes, particularly IGF2, PLAGL1, MEST, and PEG3 are overexpressed in some embryonal cancers, especially rhabdomyosarcoma, relative to non-embryonal cancers and to normal tissues. Relative overexpression in rhabdomyosarcoma cell lines compared to non-embryonal cancer cell lines was confirmed by real-time PCR for MEST, PEG3, and IGF2. For the analysis, tumors had to be classified as either embryonal or non-embryonal in origin. We classified Ewing’s sarcoma as a non-embryonal tumor because recent molecular studies suggest that these tumors are derived from the mesenchymal stem cell (36), which is an adult stem cell present in marrow rather than from an embryonal cell. Previous studies have focused primarily on IGF2, which has been shown to be overexpressed in some rhabdomyosarcoms (37–40), Wilms’ tumors (41, 42), and hepatoblastomas (43). Our findings also confirm and extend a previous report that MEST, PEG3, and DLK1 are overexpressed in Wilms’ tumor, indicating that this overexpression occurs in other embryonal cancers as well (41).

We also found that inhibiting expression of MEST, PEG3, PLAGL1, and IGF2 slowed proliferation of rhabdomyosarcoma cells in culture. In this experiment, we used siRNA to knockdown gene expression and observed significant decreases in cell proliferation, as assessed by tritiated thymidine incorporation. Two lines of evidence suggest that the decreases in proliferation were actually mediated by decreased expression of the target gene. First, using real-time RT-PCR, we demonstrated that siRNA transfection did knockdown the target mRNA levels by approximately 50–80%. Second, for each gene, we used two independent siRNAs and observed that both inhibited proliferation. It would be unlikely that these two siRNAs, which have different sequences, would both have off-target effects causing decreased proliferation. The results of the siRNA knockdown experiment indicate that MEST, PEG3, PLAGL1, and IGF2 are all required for the rapid proliferation observed in these cells, and suggests that the observed overexpression of these genes in rhabdomyosarcomas, contributes to their rapid neoplastic proliferation.

Prior studies suggests that MEST, PEG3, PLAGL1, and IGF2 are normally expressed at high levels in embryonic and early postnatal life, promoting the normal rapid body growth that occurs at this time but are subsequently down-regulated in multiple tissues simultaneously, contributing to normal somatic growth deceleration (6, 7). Thus, the current findings suggest that this downregulation fails to occur in some embryonal cancers and that this failure contributes to the persistent rapid proliferation of rhabdomyosarcoma cells and possibly their persistent embryonal characteristics.

Previous evidence also suggests that MEST, PEG3, PLAGL1, and IGF2 are members of an imprinted co-regulated gene network (13). Consistent with this proposal, we found that MEST, PEG3, and PLAGL1 expression correlated with IGF2 expression among rhabdomyosarcoma samples. Thus, the data suggest that these genes are regulated coordinately by a common mechanism. Varrault et al. have proposed that PLAGL1 may be a master regulator of this imprinted gene network (13). However, we found that siRNA-mediated knockdown of PLAGL1 expression in a rhabdomyosarcoma cell line did not consistently alter expression of MEST, PEG3, and IGF2. Therefore it is unlikely that this program of declining gene expression is coordinated by PLAGL1 in rhabdomyosarcoma cells. We also tested for other possible interactions among MEST, PEG3, PLAGL1, and IGF2 expression levels, and found only that knockdown of PEG3 decreased expression of PLAGL1. Therefore the findings suggest that the concordant regulation is not coordinated by any of these genes.

In conclusion, our findings provide evidence that, in addition to IGF2, other imprinted genes, including MEST, PEG3, and PLAGL1 are overexpressed in embryonal tumors including rhabdomyosarcomas and that this overexpression promotes growth of rhabdomyosarcoma cells. Our previous studies suggest that these genes are also highly expressed in embryonic cells and that the subsequent decline in expression contributes to normal growth deceleration. Therefore, taken together, the findings suggest that the postnatal program of gene expression that normally limits proliferation in juvenile tissues fails to occur in some embryonal cancers and contributes to the persistent rapid proliferation and perhaps embryonic characteristics of at least one embryonal cancer, rhabdomyosarcoma. Thus, MEST, PEG3, PLAGL1, IGF2, and DLK1 may provide a link between the genetic mechanisms responsible for normal growth deceleration and for the pathologic proliferation of cells in embryonal tumors. Because the imprinted genes that we studied appear to be part of a larger genetic program responsible for growth deceleration, our findings raise the possibility that failure of other components of this program may also contribute to growth of rhabdomyosarcoma, and possibly other embryonal cancers.

Acknowledgments

Financial Support: This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH.

The authors thank Dr. Javed Khan and Dr. Lee Helman of the Pediatric Oncology Branch, NCI, for helpful advice on the experimental approach and for providing cell lines.

Footnotes

Declaration of Interests: The authors have nothing to disclose

References

  • 1.Winick M, Noble A. Quantitative changes in DNA, RNA, and protein during prenatal and postnatal growth in the rat. Dev Biol. 1965;12:451–466. doi: 10.1016/0012-1606(65)90009-6. [DOI] [PubMed] [Google Scholar]
  • 2.Chailler P, Briere N. Integration of proliferation and differentiation phenomena during rodent kidney ontogeny. Growth Dev Aging. 1991;55:11–18. [PubMed] [Google Scholar]
  • 3.Post J, Hoffman J. Changes in the replication times and patterns of the liver cell during the life of the rat. Exp Cell Res. 1964;36:111–123. doi: 10.1016/0014-4827(64)90165-x. [DOI] [PubMed] [Google Scholar]
  • 4.Chang M, Parker EA, Muller TJ, Haenen C, Mistry M, Finkielstain GP, Murphy-Ryan M, Barnes KM, Sundaram R, Baron J. Changes in cell-cycle kinetics responsible for limiting somatic growth in mice. Pediatr Res. 2008;64:240–245. doi: 10.1203/PDR.0b013e318180e47a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Finkielstain GP, Forcinito P, Lui JC, Barnes KM, Marino R, Makaroun S, Nguyen V, Lazarus JE, Nilsson O, Baron J. An extensive genetic program occurring during postnatal growth in multiple tissues. Endocrinology. 2009;150:1791–1800. doi: 10.1210/en.2008-0868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Lui JC, Forcinito P, Chang M, Chen W, Barnes KM, Baron J. Coordinated postnatal down-regulation of multiple growth-promoting genes: evidence for a genetic program limiting organ growth. FASEB J. 2010;24:3083–3092. doi: 10.1096/fj.09-152835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Lui JC, Finkielstain GP, Barnes KM, Baron J. An imprinted gene network that controls mammalian somatic growth is down-regulated during postnatal growth deceleration in multiple organs. Am J Physiol Regul Integr Comp Physiol. 2008;295:R189–196. doi: 10.1152/ajpregu.00182.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.DeChiara TM, Efstratiadis A, Robertson EJ. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature. 1990;345:78–80. doi: 10.1038/345078a0. [DOI] [PubMed] [Google Scholar]
  • 9.Lefebvre L, Viville S, Barton SC, Ishino F, Keverne EB, Surani MA. Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest. Nat Genet. 1998;20:163–169. doi: 10.1038/2464. [DOI] [PubMed] [Google Scholar]
  • 10.Li L, Keverne EB, Aparicio SA, Ishino F, Barton SC, Surani MA. Regulation of maternal behavior and offspring growth by paternally expressed Peg3. Science. 1999;284:330–333. doi: 10.1126/science.284.5412.330. [DOI] [PubMed] [Google Scholar]
  • 11.Gerard M, Hernandez L, Wevrick R, Stewart CL. Disruption of the mouse necdin gene results in early post-natal lethality. Nat Genet. 1999;23:199–202. doi: 10.1038/13828. [DOI] [PubMed] [Google Scholar]
  • 12.Van Dyck F, Declercq J, Braem CV, Van de Ven WJ. PLAG1, the prototype of the PLAG gene family: versatility in tumour development (review) Int J Oncol. 2007;30:765–774. [PubMed] [Google Scholar]
  • 13.Varrault A, Gueydan C, Delalbre A, Bellmann A, Houssami S, Aknin C, Severac D, Chotard L, Kahli M, Le Digarcher A, Pavlidis P, Journot L. Zac1 regulates an imprinted gene network critically involved in the control of embryonic growth. Dev Cell. 2006;11:711–722. doi: 10.1016/j.devcel.2006.09.003. [DOI] [PubMed] [Google Scholar]
  • 14.Deng Y, Wu X. Peg3/Pw1 promotes p53-mediated apoptosis by inducing Bax translocation from cytosol to mitochondria. Proc Natl Acad Sci U S A. 2000;97:12050–12055. doi: 10.1073/pnas.97.22.12050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Wood AJ, Oakey RJ. Genomic imprinting in mammals: emerging themes and established theories. PLoS Genet. 2006;2:e147. doi: 10.1371/journal.pgen.0020147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Paulino AC, Okcu MF. Rhabdomyosarcoma. Curr Probl Cancer. 2008;32:7–34. doi: 10.1016/j.currproblcancer.2007.11.001. [DOI] [PubMed] [Google Scholar]
  • 17.Whiteford CC, Bilke S, Greer BT, Chen Q, Braunschweig TA, Cenacchi N, Wei JS, Smith MA, Houghton P, Morton C, Reynolds CP, Lock R, Gorlick R, Khanna C, Thiele CJ, Takikita M, Catchpoole D, Hewitt SM, Khan J. Credentialing preclinical pediatric xenograft models using gene expression and tissue microarray analysis. Cancer Res. 2007;67:32–40. doi: 10.1158/0008-5472.CAN-06-0610. [DOI] [PubMed] [Google Scholar]
  • 18.Hosoi H, Dilling MB, Liu LN, Danks MK, Shikata T, Sekulic A, Abraham RT, Lawrence JC, Jr, Houghton PJ. Studies on the mechanism of resistance to rapamycin in human cancer cells. Mol Pharmacol. 1998;54:815–824. doi: 10.1124/mol.54.5.815. [DOI] [PubMed] [Google Scholar]
  • 19.Cocker HA, Tiffin N, Pritchard-Jones K, Pinkerton CR, Kelland LR. In vitro prevention of the emergence of multidrug resistance in a pediatric rhabdomyosarcoma cell line. Clin Cancer Res. 2001;7:3193–3198. [PubMed] [Google Scholar]
  • 20.Onisto M, Slongo ML, Gregnanin L, Gastaldi T, Carli M, Rosolen A. Expression and activity of vascular endothelial growth factor and metalloproteinases in alveolar and embryonal rhabdomyosarcoma cell lines. Int J Oncol. 2005;27:791–798. [PubMed] [Google Scholar]
  • 21.Stratton MR, Reeves BR, Cooper CS. Misidentified cell. Nature. 1989;337:311–312. doi: 10.1038/337311c0. [DOI] [PubMed] [Google Scholar]
  • 22.McAllister RM, Melnyk J, Finkelstein JZ, Adams EC, Jr, Gardner MB. Cultivation in vitro of cells derived from a human rhabdomyosarcoma. Cancer. 1969;24:520–526. doi: 10.1002/1097-0142(196909)24:3<520::aid-cncr2820240313>3.0.co;2-m. [DOI] [PubMed] [Google Scholar]
  • 23.Chen TR. Re-evaluation of HeLa, HeLa S3, and HEp-2 karyotypes. Cytogenet Cell Genet. 1988;48:19–24. doi: 10.1159/000132579. [DOI] [PubMed] [Google Scholar]
  • 24.Chakrabarty S, Bratain MG. The use of 125I-lectin probes in defining plasma membrane carbohydrate moieties in 3 subpopulations of human colonic carcinoma cells. Cancer Lett. 1987;37:99–108. doi: 10.1016/0304-3835(87)90150-9. [DOI] [PubMed] [Google Scholar]
  • 25.Lasfargues EY, Coutinho WG, Redfield ES. Isolation of two human tumor epithelial cell lines from solid breast carcinomas. J Natl Cancer Inst. 1978;61:967–978. [PubMed] [Google Scholar]
  • 26.Littlewood-Evans AJ, Bilbe G, Bowler WB, Farley D, Wlodarski B, Kokubo T, Inaoka T, Sloane J, Evans DB, Gallagher JA. The osteoclast-associated protease cathepsin K is expressed in human breast carcinoma. Cancer Res. 1997;57:5386–5390. [PubMed] [Google Scholar]
  • 27.Scotlandi K, Avnet S, Benini S, Manara MC, Serra M, Cerisano V, Perdichizzi S, Lollini PL, De Giovanni C, Landuzzi L, Picci P. Expression of an IGF-I receptor dominant negative mutant induces apoptosis, inhibits tumorigenesis and enhances chemosensitivity in Ewing’s sarcoma cells. Int J Cancer. 2002;101:11–16. doi: 10.1002/ijc.10537. [DOI] [PubMed] [Google Scholar]
  • 28.Hofbauer S, Hamilton G, Theyer G, Wollmann K, Gabor F. Insulin-like growth factor-I-dependent growth and in vitro chemosensitivity of Ewing’s sarcoma and peripheral primitive neuroectodermal tumour cell lines. Eur J Cancer. 1993;29A:241–245. doi: 10.1016/0959-8049(93)90183-g. [DOI] [PubMed] [Google Scholar]
  • 29.Smith MA, Morton CL, Phelps D, Girtman K, Neale G, Houghton PJ. SK-NEP-1 and Rh1 are Ewing family tumor lines. Pediatr Blood Cancer. 2008;50:703–706. doi: 10.1002/pbc.21099. [DOI] [PubMed] [Google Scholar]
  • 30.Heldin CH, Johnsson A, Wennergren S, Wernstedt C, Betsholtz C, Westermark B. A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A-chains. Nature. 1986;319:511–514. doi: 10.1038/319511a0. [DOI] [PubMed] [Google Scholar]
  • 31.Raile K, Hoflich A, Kessler U, Yang Y, Pfuender M, Blum WF, Kolb H, Schwarz HP, Kiess W. Human osteosarcoma (U-2 OS) cells express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors and synthesize IGF-II: autocrine growth stimulation by IGF-II via the IGF-I receptor. J Cell Physiol. 1994;159:531–541. doi: 10.1002/jcp.1041590317. [DOI] [PubMed] [Google Scholar]
  • 32.Fogh J, Wright WC, Loveless JD. Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J Natl Cancer Inst. 1977;58:209–214. doi: 10.1093/jnci/58.2.209. [DOI] [PubMed] [Google Scholar]
  • 33.Huber C, Sundstrom C, Nilsson K, Wigzell H. Surface receptors on human haematopoietic cell lines. Clin Exp Immunol. 1976;25:367–376. [PMC free article] [PubMed] [Google Scholar]
  • 34.Lozzio BB, Lozzio CB. Properties and usefulness of the original K-562 human myelogenous leukemia cell line. Leuk Res. 1979;3:363–370. doi: 10.1016/0145-2126(79)90033-x. [DOI] [PubMed] [Google Scholar]
  • 35.Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, Nagura H, Saito H. Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. Blood. 1985;66:1384–1392. [PubMed] [Google Scholar]
  • 36.Ewing Jedlicka P. Sarcoma, an enigmatic malignancy of likely progenitor cell origin, driven by transcription factor oncogenic fusions. Int J Clin Exp Pathol. 3:338–347. [PMC free article] [PubMed] [Google Scholar]
  • 37.Anderson J, Gordon A, McManus A, Shipley J, Pritchard-Jones K. Disruption of imprinted genes at chromosome region 11p15.5 in paediatric rhabdomyosarcoma. Neoplasia. 1999;1:340–348. doi: 10.1038/sj.neo.7900052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Anderson J, Gordon A, Pritchard-Jones K, Shipley J. Genes, chromosomes, and rhabdomyosarcoma. Genes Chromosomes Cancer. 1999;26:275–285. [PubMed] [Google Scholar]
  • 39.Rikhof B, de Jong S, Suurmeijer AJ, Meijer C, van der Graaf WT. The insulin-like growth factor system and sarcomas. J Pathol. 2009;217:469–482. doi: 10.1002/path.2499. [DOI] [PubMed] [Google Scholar]
  • 40.Wada M, Seeger RC, Mizoguchi H, Koeffler HP. Maintenance of normal imprinting of H19 and IGF2 genes in neuroblastoma. Cancer Res. 1995;55:3386–3388. [PubMed] [Google Scholar]
  • 41.Dekel B, Metsuyanim S, Schmidt-Ott KM, Fridman E, Jacob-Hirsch J, Simon A, Pinthus J, Mor Y, Barasch J, Amariglio N, Reisner Y, Kaminski N, Rechavi G. Multiple imprinted and stemness genes provide a link between normal and tumor progenitor cells of the developing human kidney. Cancer Res. 2006;66:6040–6049. doi: 10.1158/0008-5472.CAN-05-4528. [DOI] [PubMed] [Google Scholar]
  • 42.Algar EM, St Heaps L, Darmanian A, Dagar V, Prawitt D, Peters GB, Collins F. Paternally inherited submicroscopic duplication at 11p15.5 implicates insulin-like growth factor II in overgrowth and Wilms’ tumorigenesis. Cancer Res. 2007;67:2360–2365. doi: 10.1158/0008-5472.CAN-06-3383. [DOI] [PubMed] [Google Scholar]
  • 43.Kim KS, Lee YI. Biallelic expression of the H19 and IGF2 genes in hepatocellular carcinoma. Cancer Lett. 1997;119:143–148. doi: 10.1016/s0304-3835(97)00264-4. [DOI] [PubMed] [Google Scholar]

RESOURCES