Loss of p21 permits carcinogenesis from chronically damaged liver and kidney epithelial cells despite unchecked apoptosis

Abstract

Accumulation of toxic metabolites in hereditary tyrosinemia type I (HT1) patients leads to chronic DNA damage and the highest risk for hepatocellular carcinomas (HCCs) of any human disease. Here we show that hepatocytes of HT1 mice exhibit a profound cell-cycle arrest that, despite concomitant apoptosis resistance, causes mortality from impaired liver regeneration. However, additional loss of p21 in HT1 mice restores the proliferative capabilities of hepatocytes and renal proximal tubular cells. This growth response compensates cell loss due to uninhibited apoptosis and enables animal survival but rapidly leads to HCCs, renal cysts, and renal carcinomas. Thus, p21's antiproliferative function is indispensable for the suppression of carcinogenesis from chronically injured liver and renal epithelial cells and cannot be compensated by apoptosis.

Figure 1. Fah Deficiency Causes Cell Cycle Arrest Associated with DNA Damage and Induction of p21

(A) In contrast to wild-type cells (upper panel), Fah-deficient hepatocytes (lower panel) refrain from entry into S-phase in response to PH as evident from lack of BrdU labeling (brown, arrowhead in lower panel). Scale bars indicate 100 μm. (B) p21 but not p27 protein is induced in Fah^−/− mice off NTBC to levels markedly exceeding those associated with the physiological halt of liver regeneration 72 hours after PH (H) in Fah^−/− mice on NTBC. p21 protein levels are maximally induced in Fah^−/− mice off NTBC for 14 days since they fail to increase after further stimulation by PH. (C) Comet assay shows that NTBC withdrawal (grey columns) causes DNA damage (tail moment) in Fah^−/− and Fah^−/−, p21^−/− livers. Fah^−/−, p21^−/− mice exhibit increased levels of DNA damage both on (white columns) and off NTBC. Error bars represent mean ± standard deviation. (D) DNA damage in Fah^−/− and even more so in Fah^−/−, p21^−/−hepatocytes is reflected by induction of p53. (E and F) p21 protein induced by Fah deficiency is distributed between cytoplasm and nucleus as evident from comparison by Western Blot to c-jun which is strictly limited to the nucleus (E) and p21 immunocytochemistry (brown, left panel) on plated hepatocytes costained for DNA with SYBR Green I (green, right panel) (F). Inset shows absence of p21 in Fah^−/−, p21^−/− hepatocyte (F, left panel). Scale bars indicate 25 μm.

Figure 2. p21 Represses the Regenerative Response of Hepatocytes to Cell Damage Inflicted by Fah Deficiency

(A) Wild-type liver contains only very few proliferating (brown, BrdU labeling) hepatocytes (left panel, arrowhead). Upon NTBC withdrawal, Fah^−/− hepatocytes (middle panel) fail to enter the S phase of the cell cycle whereas Fah^−/−, p21^−/− hepatocytes (right panel) show massive proliferation. Note dysplasia of Fah^−/− and Fah^−/−, p21^−/− hepatocytes off NTBC compared to wild-type cells. (B and C) Immunohistochemistries (brown staining) for Ki67 (B) and phosphorylated histone H3 (C) show that despite liver injury induced by NTBC withdrawal Fah^−/− hepatocytes (middle panels) exhibit a proliferation arrest similar to wild-type cells (arrowheads, left panels) which is lifted in livers deficient in both Fah and p21 (right panels). Progression of Fah^−/−, p21^−/− hepatocytes into the G2 and M phases of the cell cycle is apparent from both Ki67 (B, right panel) and phosphorylated histone H3 (C, right panel) staining. Quantification of Ki67-labeled hepatocytes is provided in Supplemental Figure 2. Scale bars indicate 100 μm. (D) Kaplan-Meier plot shows that Fah^−/−, p21^−/− mice (blue line) but not their Fah^−/− littermates (red line) survive beyond the critical phase of NTBC withdrawal. 10 mice were analyzed for each genotype. (E) The cell cycle-inhibitory function of p21 in hepatocytes is dose-dependent. 14 days after NTBC withdrawal p21 protein levels are maximally induced. These p21 protein levels are needed for suppression of hepatocyte proliferation in Fah^−/− mice after PH as levels of p21 protein found after 4 or 8 days off NTBC fail to prevent liver regeneration (data not shown). 3 mice were analyzed for each time point and a representative Western Blot is shown. α-actin serves as loading control.

Figure 3. Apoptosis Resistance Inherent to Fah Deficiency Depends on p21

(A and B) In contrast to wild-type (left panels) or Fah^−/− (middle panels) mice, Fah^−/−, p21^−/− (right panels) animals exhibit increased numbers of TUNEL positive hepatocytes (brown, arrowheads) after 2 weeks off NTBC (A) which can be further increased by application of Fas (B). (C) Hepatic apoptosis response in Fah-deficient mice depends on p21 and NTBC status. Spontaneous caspase-3 activation is detectable in liver lysates from Fah^−/−, p21^−/− mice off NTBC for 4 weeks (5th column) but not 2 weeks (4th column). In contrast, caspase-3 activation is undetectable in liver lysates from Fah^−/−, p21^−/− and Fah^−/− mice on NTBC (3rd and 1st column) or Fah^−/− mice after 2 or 4 weeks of NTBC withdrawal (2nd column). Fas injection induces massive caspase-3 activation in both Fah^−/− and Fah^−/−, p21^−/− mice on NTBC (6th and 8th column). Fah^−/− mice off NTBC show resistance to Fas-induced apoptosis (7th column) which is abrogated in Fah^−/−, p21^−/−mice off NTBC (9th column). Error bars represent mean ± standard deviation. (D) Quantification of the percentage of TUNEL positive hepatocytes in untreated (dark grey columns) and Fas-injected (light grey columns) mice. (E) Injection of Fas is followed by BrdU labeling (brown staining) of many Fah^−/−, p21^−/− hepatocytes (right panel) while only very few Fah^−/− hepatocytes (left panel, arrowhead) exhibit compensatory proliferation. 3 mice were analyzed for each genotype and treatment regimen. Representative stainings are shown and corresponding H&E stainings are provided in Supplemental Figure 3. Scale bars indicate 100 μm.

Figure 4. DNA-Damaged Hepatocytes Give Rise to Liver Cancer in the Absence of p21

(A to C) AFP immunohistochemistry (brown staining) indicates that wild-type livers (A) lack dysplastic hepatocytes while similar numbers of these cells are present in Fah^−/− (B) and Fah^−/−, p21^−/− (C) mice off NTBC. Scale bars indicate 100 μm. (D) Ki67 immunohistochemistry (brown staining) marking proliferation of dysplastic hepatocytes in a Fah^−/−, p21^−/− mouse off NTBC. Scale bar indicates 50 μm. (E) Representative H&E staining of WHO grade 3 HCC in Fah^−/−, p21^−/− mouse off NTBC. Scale bar indicates 50 μm. (F) Median lobe of Fah^−/−, p21^−/− mouse off NTBC after clonal progression of dysplastic hepatocytes to cancer nodules.

Figure 5. Unrestricted Proliferation of DNA-Damaged Renal Proximal Tubular Cells Results in Formation of Cysts and Carcinomas

(A and B) Fah^−/−, p21^−/− mice develop renal cysts after NTBC withdrawal. (A) Large cysts extend to the surface of the kidney (scale in inches). (B) Cystically dilated proximal convoluted tubules and cysts. Scale bar indicates 200 μm. (C to F) Progression of renal tubular dysplasia to renal carcinomas in Fah^−/−, p21^−/− mice off NTBC. (C) Dysplastic renal proximal tubular cells with large and hyperchromatic nuclei. Scale bar indicates 50 μm. (D) Immunohistochemistry for Ki67 (brown staining) shows cluster of proliferating dysplastic renal epithelial cells. Scale bar indicates 50 μm. (E) Kidney cancer nodule compressing the surrounding parenchyma. Scale bar indicates 100 μm. (F) Representative H&E staining of grade 2–3 (Fuhrman classification) renal cell carcinoma. Scale bar indicates 50 μm.