c-Myc regulates cell size and ploidy but is not essential for postnatal proliferation in liver

Abstract

The c-Myc protein is a transcription factor implicated in the regulation of multiple biological processes, including cell proliferation, cell growth, and apoptosis. In vivo overexpression of c-myc is linked to tumor development in a number of mouse models. Here, we show that perinatal inactivation of c-Myc in liver causes disorganized organ architecture, decreased hepatocyte size, and cell ploidy. Furthermore, c-Myc appears to have distinct roles in proliferation in liver. Thus, postnatal hepatocyte proliferation does not require c-Myc, whereas it is necessary for liver regeneration in adult mice. These results show novel physiological functions of c-myc in liver development and hepatocyte proliferation and growth.

Fig. 1.

Altered liver organization in homozygous c-myc^fl/fl;mx-cre⁺ mice. (A) Deletion of c-myc gene in homozygous c-myc^fl/fl;mx-cre⁺ mice. Genomic DNA from the organs shown was isolated, EcoRI-digested, and probed as described (24). Two perinatally pIpC-injected 10-week-old homozygous c-myc^fl/fl;mx-cre⁺ mice are shown. (B) Deletion of c-myc gene in c-myc^fl/fl;mx-cre⁺ mouse liver. Genomic DNA from pIpC-injected 10 day-old and 10 week-old c-myc^fl/fl;mx-cre⁺ mice was used. Southern blot was performed as in A. Numbers indicate individual mice. Genomic DNA from c-myc^fl/fl;mx-cre^- mice or wt mice was used as a control. Experiment representative of multiple experiments. (C) Quantification of flox and null alleles in livers from pIpC-injected 10 day-old and 10-week-old homozygous c-myc^fl/fl;mx-cre⁺ mice. Quantification was performed with PhosphorImager, using imagequant software on Southern blots performed as in B. %A allele, A allele counts × 100/total alleles counts (flox plus null counts). A is either null or flox alleles. (D) H&E-stained sections from 10 day-old and 10-week-old homozygous c-myc^fl/fl;mx-cre⁺ liver and control mouse liver. Seven 10-day-old mice of each genotype and seven 10-week-old mice of each genotype were analyzed. (E) Liver:body weight ratio of 10-week-old homozygous c-myc^fl/fl;mx-cre⁺ and control mice. (F) Body weight of 10-week-old homozygous c-myc^fl/fl;mx-cre⁺ mice and control mice from E (n = 30 for each genotype). (G) Hepatic function in 10-week-old mice. Serum levels of aspartate (AST/GOT) and alanine (ALT/GPT) aminotransferases. n = 14 for homozygous mice and n = 6 for control mice. U/L, units per liter. ^*, P = 0.0202; ^**, P = 0.0327 (Student's t test). In all experiments, mice were injected with pIpC as newborns (see Materials and Methods).

Fig. 2.

Decreased cell size and ploidy in hepatocytes from c-myc^fl/fl; mx-cre⁺mice. (A) Reduced hepatocyte size in c-myc^fl/fl;mx-cre⁺ mice. Hepatocytes from 10-week-old homozygous c-myc^fl/fl;mx-cre⁺ mice and c-myc^fl/fl;mx-cre^- control mice were isolated and analyzed by FACS. A total of four mice of each genotype were analyzed. (B) Area of hepatocytes from H&E-stained liver sections of 10-week-old mice (imagej29 software). pX², square pixels. (C) Hepatocyte number per field (two fields per mouse) in liver sections from mice as in Fig. 1D. For cell area and hepatocytes per field, five control mice and nine homozygous mice were analyzed. (D) Decreased cell ploidy in hepatocytes from 10-week-old homozygous c-myc^fl/fl;mx-cre⁺ mice. Side-scatter plot of whole populations is shown. R1 gate is used to discriminate polyploid cells; R2 gate contains nonpolyploid cells only. Numbers represent the percentage of cells in each gate within each plot. (E) Cell size vs. DNA content (propidium iodide). Hepatocytes from mice of indicated genotypes were isolated and analyzed by FACS. All mice were injected with pIpC as newborns. A line has been drawn to compare FSC values. Example representative of three independent experiments. (F) Reduced number of binucleated cells in liver from c-myc^fl/fl;mx-cre⁺ mice. The number of binucleated cells per field were counted (three fields per mouse), and the number was normalized by 100 cells per field. The graph shows the mean of four mice for each genotype. P value was determined by using Student's t test.

Fig. 3.

Hepatocyte proliferation in the absence of c-Myc. (A) Proliferation of hepatocytes from 6- and 10-week-old homozygous c-myc^fl/fl;mx-cre⁺ mice and c-myc^fl/fl;mx-cre^- control mice. BrdUrd was added to drinking water for 1 week. Liver sections were prepared and stained with an anti-BrdUrd antibody. Arrows indicate hepatocytes (H) or lymphocytes (L). Two control mice and three 6- or 10-week-old homozygous mice were analyzed. (B) Genomic PCR of single PCNA⁺cells from c-myc^fl/fl;mx-cre⁺ mice. Liver sections were prepared and stained with anti-PCNA antibody. Single PCNA⁺ cells were isolated by laser dissection. Genomic DNA from each single PCNA⁺ cell was PCR-amplified by using flox (nondeleted)- and null (deleted)-specific primers (see Materials and Methods). Genomic DNA from tail was used as a positive control (+C). Eleven PCNA⁺ cells from three homozygous c-myc^fl/fl;mx-cre⁺ mice were analyzed in two independent experiments. (C) Impaired liver regeneration in homozygous c-myc^fl/fl;mx-cre⁺ mice injected as adults. Six-week-old mice were injected with pIpC. PH was performed; 48 h later, mice were killed and protein from liver was prepared. PCNA and cyclin A were measured by Western blot. Actin was used as a loading control. Three homozygous c-myc^fl/fl;mx-cre⁺ mice and two control mice were analyzed. (D) RPA with total RNA (10 μg) from liver of 10-day-old homozygous c-myc^fl/fl;mx-cre⁺ mice and control mice. Three mice of each genotype are shown. Experiment representative of two independent experiments. In all experiments, except for the experiment with liver regeneration, mice were injected with pIpC as newborns.

Fig. 4.

Apoptosis in liver from c-myc^fl/fl;mx-cre⁺. (A) TUNEL assays in liver sections from homozygous c-myc^fl/fl;mx-cre⁺ mice and control mice. (B) Increased free-radical content in homozygous c-myc^fl/fl;mx-cre⁺ mouse liver. Hepatocytes from 10-day-old and 10-week-old homozygous and control mice were isolated and stained with the oxidation-sensitive probe DCFH-DA; a total of four mice of each genotype were analyzed. (C) Hepatocyte apoptosis is inhibited by the antioxidant l-ascorbic acid (L-Asc). Hepatocytes from c-myc^fl/fl;mx-cre⁺ mice and control mice were isolated and cultured in vitro alone or with L-Asc for 24 h. Cells were harvested at times indicated, propidium iodide-stained to measure DNA content, and subG₀/G₁ peaks (dead cells) were analyzed by FACS. Experiment representative of three independent experiments. In all experiments, mice were injected with pIpC as newborns.