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Receptor targeting to hepatocytes increases polyethylenimine-mediated nonviral delivery of nucleic acids to the liver
384A
AASLD ABSTRACTS
HEPATOLOGY O c t o b e r 2 0 0 1
847
848
RECEPTOR TARGETING TO HEPATOCYTES INCREASES POLYETHYLE N I M I N E - M E D I A T E D NONVIRAL DELIVERY OF NUCLEIC ACIDS TO THE LIVER. Paul D Richardson, Rod Felsheim, Gladys Chuey, Betsy T Kren, Clifford J Steer, U n i v e r s i t y of Minnesota Medical School, Minneapolis, M N
O S T E O A C T M N , A GENE ISOLATED FROM THE LIVER OF RAT FED W I T H A CHOLINE-DEFICIENT, L-AMINO ACID DEFINED DIET, ACCELERATED INVASION AND METASTASIS OF tIEPATOMA CELLS. Masaaki Onaga, Akio Ido, Akihiro Moriuti, Yukiko O n a g a , Satoru Hasnike, H i r o f u m i Uto, Kenji Nagata, Takeshi Hori, Shuichi Hirono, Katsuhiro Hayashi, Hirohito T s u b o u c h i , Miyazaki Medical College, Kiyotake T o w n J a p a n
There are now well-defined fundamental aspects of any successful gene therapy/targeted gene repair process. These include efficient packaging of DNA; effective delivery and cell specificity with minimal toxicity-; and efficient nuclear translocation. The liver is an attractive organ for gene therapy because it is central to many inherited and acquired genetic disorders. In addition, a large blood supply enables efficient delivery and extravasation of complexes through fenestrae. The asialoglycoprotein receptor is unique to hepatocytes and highly expressed on the cell surface membrane. Polyethylenimine (PEI) is a cationic polymer able to condense DNA and afford adequateprotection against nuelsases. PEI possesses a large number of uncharged amines that enable it to act as a proton sponge after cellular uptake, preventin~ vesicle acidification, inhibiting intracellular nucleases and allowing osmotic rupture ot the endosomes. In addition, PEI may also possess nuclear localizing properties. Unlike some other polycations, PEI is an effective agent in serum, which is obviously critical for in vivo delivery. Lactosylation of the PEI enables targeting to the asialoglycoprotein receptor and hence mediates hepatocyte specificity. The AIM of these studies was to optimize the delivery of both the iuciferase reporter plasmid pGL3 and florescently-labeled single-stranded oligonucleotides (SSOs) to human hepatoma HUH-7 cells, primary rat hepatocytes and primary mouse fibroblasts. We have recently shown that SSOs are effective mediators of site-specific gene repair when delivered in high copy number to cells. METHODS: Branched 10 kDa and linear and branched 25 kDa formulations of PEI were used. Lactose was covalentiy attached to either the primary or secondary amines on the branched PEIs by EDAC conjugation or Schiff-base reaction with titanium isopropoxide, respectively. The efficacy of delivery for each of these different formulations was compared using a reporter plasmid encoding lucfferase and a Cy-3-1abeled 35 mer SSO. The relative sizes of the PEI:DNA complexes were examined by transmission electron microscopy (TEM), and nuclear delivery assessed by confocal microscopy. Luciferase activity was determined by a luminometric assay. RESULTS: Delivery of the plasmid was most efficient with the branched 25 kDa PEI resulting in luciferase activity > 10 t° RLU/mg protein. This was an order of magnitude greater than the branched 10 kDa or linear 25 kDa PEI, but was generally more toxic. Lactosylation of the PEI resulted in a marked increase in uptake by hepatocytes, which was competitively blocked by coincubation with EGTA or D-galactose. In contrast, no significant changes in uptake were Observed with fibroblasts. Primary amine lactosylation increased the delivery efficiency by at least 2 orders of magnitude over secondary amine conjugation. Linear PEI was most effective in delivering SSOs and resulted in a more rapid and significantly increased nuclear localization relative to the branched PEIs. In fact, there was marked nuclear uptake within 1 hour of transfection. Under all conditions, delivery was significantly reduced in primary rat hepatocytes compared to HUH-7 cells. TEM of the 25 kDa 7 kb plasmid complexes were < 50 nm in size and monodisperse. Interestingly, the branched 10/25 kDa combination exhibited larger complexes of ~ 65 nm. The linear complexes tended to aggregate and were typically > 100 nm in size. CONCLUSIONS: PEI is an effective agent in delivering both plasmids and small nucleic acids to hepatocytes and primary fibroblasts. The optimal formulation of PEI was clearly influenced by the relative size of the nucleic acid molecule delivered to the cell. Lactosylation of PEI provides a simple modification to significantly improve specificity and uptake by hepatocytes. The PEI polycatinn remains a versatile and potentially effective agent in the delivery of nucleic acids for gene therapy.
BACKGROUND: The development of hepatocellular carcinoma (HCC) is associated with chronic liver disease, particularly cirrhosis. However, little is known about the genes involved in hepatocarcinogenesis associated with cirrhosis. In a rat model of hepatocarcinogenesis induced by a choline-deficient, L-amino acid defined (CDAA) diet, initial steatosis and subsequent cirrhosis is followed by occurrence of HCC. In this study, to clarify the genes associated with hepatocarcinogenesis at an early phase, we examined the genes expressed in the liver of rats fed a CDAA diet for 2 months and isolated osteoactivin (OA) cDNA. We also analyzed the OA expression in human HCC tissues and the effect of OA on invasion and metastasis of hepatoma cells. Materials and Methods: (1) The genes expressed in the liver of rats fed with a CDAA diet for 2 months were isolated using suppression subtractive hybridization. (2) Northern blot analysis was performed to examine the expression of OA mRNA in the liver of rats fed with a CDAA diet, human HCC and normal liver tissues. (3) OA- or aatisense-OA cDNA was introduced into rat hepatoma cells (dRLh84), and G418-resistant clorlal cell lines (dRLh84/OA or dRLh84/AS-OA, respec~ tively) were established. (4) The effect of OA on invasiveness in the presence or absence of hepatocyte growth factor (HGF) was examined by Matrigel invasion assays. (5) To analyze the OA-mediated metastasis, dRLH84/parent and dRLH84/OA were inoculated into the spleen of nude mice, and the development of hepatic metastasis was evaluated. RESULTS: (1) We isolated transferrin, NF-KB, uncoupling protein-2 and OA cDNAs. (2) OA mRNA was strongly expressed in the liver of rat fed with a CDAA diet for 1 to 3 months and moderate expression continued until 18 months. Also, in the HCC developing in the rat given a CDAA diet for 18 months, a higher level of OA mRNA expression was observed. In humans, OA mRNA expression was detected in both HCC and the tissues surrounding the lesion, whereas OA was not expressed in normal liver. The level of OA expression in well-differentiated HCCs was higher than that in the surrounding liver tissues as well as moderately- and poorly-differentiated HCCs. (3) The expression of OA mRNA increased twice in dRLh84/OA cells and decreased to 30% in dRLh84/AS-OA ceils, as compared with dRLh84/parent. (4) The number ofdRLh84/OA ceils invading the Matrigel membrane was 10-tbld higher than that of dRLh84/parem (p<0.05). HGF stimulated the invasiveness of dRLh84/parental cells by 50-fold. However, HGF treatment induced only a 4-fold increase in the Matrigel invasiveness of dRLh84/AS-OA ceils (p<0.001). (5) When dRLh84/parental cells were inoculated into the spleen of nude mice, metastatic liver tumors were observed in 12.5% (1/8) of mice. In contrast, intrasplenic inoculation of dRLh84/OA cells resulted in hepatic metastasis in all mice (16/16) (p<0.005). ICONCLUSIONS] We isolated OA gene from the rat liver during the early phase of a CDAA diet-induced hepatocarcinogenesis. OA was strongly expressed in not only rat HCC tissues but also human HCC tissues, and OA overexpression accerelated the invasiveness and metastasis of hepatoma ceils, These results suggest that OA is a novel factor involved in hepatocarcinogenesis and tumor progression through the stimulation of tumor invasiveness and metastasis of HCC.
849
850
FUNCTIONAL COOPERATION OF P18 INK4c W I T H P21 cwl/wAF1 AND P27 ~P1 IN THE REGENERATING LIVER. T o m Luedde, D e p a r t m e n t of Gast r o e n t e r o l o g y a n d H e p a t o l o g y , Medizinische H o c h s c h u l e H a n n o v e r , Germ a n y , H a n n o v e r G e r m a n y ; Maria E Rodriguez, L i n e b e r g e r C o m p r e h e n s i v e C a n c e r Center, U N C at Chapel Hill, NC, Chapel Hill, NC; Christian T r a u t w e i n , D e p a r t m e n t of G a s t r o e n t e r o l o g y a n d Hepatology, Medizinische H o c h s c h u l e H a n n o v e r , G e r m a n y , H a n n o v e r G e r m a n y ; David A Brenner, Dep a r t m e n t of Digestive Diseases a n d Nutrition, U N C at Chapel Hill, NC, Chapel Hill, NC; Yue Xiong, L i n e b e r g e r C o m p r e h e n s i v e C a n c e r Center, U N C at Chapel Hill, NC, C h a p e l Hill, N C
DEVELOPING LIVERS DISPLAY AN UNIQUE TRANSCRIPTIONAL PROGRAM W I T H MINIMAL OVERLAP W I T H HEPATIC REGENERATION. N a n c y Kelley-Loughnane, G r e g g Sabla, Bruce J A r o n o w , Jorge A Bezerra, Children's Hospital Medical Center, Cincinnati, O H
Entry of quiescent cells into the cell cycle is driven by the cyclin D-dependent kinases Cdk4 and Cdk6. pl8 INK4eis a member of the INK4 family of proteins that specifically target the Cdk4 and Cdk6 kinases and inhibit their catalytic activity. The ability of p 18 to control G1 phase progression has been studied in vitro, but less is known about the significance of this inhibition in vivo. As such, the role of p18 for cell cycle control in the liver is unclear. In this study, we used mice lacking p18zNK~c,p 2 t cwzAvAFl or p27KIP1alone and mice lacking both p18/p21 or p18/p27. The mice were analyzed for differences in the timing of ceil cycle progression after partial hepatectomy (PH). An IP-Western showed that in wild type (WT) mice p18 is expressed at a lower level in the quiescent liver compared to NIH 3T3 cells. After PH this basic level remained unchanged during the first 96h. In the p18 deficient mice PCNA expression as a marker for S-Phase entry and Cyclin D expression revealed no changes compared to the WT control mice with maximum expression at 48h after PH. BrdU-staining used as an additional marker for S-Phase entry of ceils showed that, like in the control group, the majority of nuclei (~50%) were positive 48h after PH and almost no positive nucleus was found after 36h. Loss of p21 function resuhed in a shorter G1 interval: Both PCNA and cycfin D1 were expressed at 36h post-PH with maximum expression at 48h post-PH. At 36h after PH about 10% of the nuclei stained positive for BrdU, but the majority of nuclei (~40%) were positive after 48h. The group of p27 mutant mice had a minimally increased level of PCNA expression prior to PH compared to WT control. However, upregulation of PCNA expression to S-Phase specific levels occurred no earlier than 48h after PH. Cyelin D expression and BrdU staining showed no changes to the WT control group. In the mice lacking both pl8 and p21 we found that the G1 Phase interval after PH was even shorter than in the p21 single knockout animals. PCNA as well as Cyclin D expression began already 24h after PH with maximum expression at 36h after PH. BrdU staining showed that the majority of nuclei (~40%) were already positive 36h post PH and only"a minor portion was still positive after 48h (~10%). Finally the plB/p27 double-knockout mice showed an even stronger increase of base level PCNA expression and a stronger maximum compared to both the WT control animals and p27 null-mice, but no changes in the timing of the expression was evident. Cyclin D expression remained unchanged in the p18/p27 double mutant animals compared to both groups. Furthermore at 36h post-PH almost no nuclei were BrdU-positive whereas 48h post-PH almost 90% of hepatocytes were BrdU-positive. Our results demonstrate for the first time that pl8, although expressed at a low level in the liver, is important for controlling cell cycle progression in proliferating hepatocytes in vivo. Moreover, our study indicates that pig collaborates with other inhibitors of cyclin dependent kinases to regulate different aspects of cell cycle progression: p 18 and p21 collaborate in regulating the length of the G1 phase after PH while pi8 and p27 seem to cooperate in modulating the threshold of bepatocytes to enter the cell cycle as disruption of these two genes leads to almost double the number of hepatocytes that synchronously enter the cell cycle after PH. These findings could also have implications for the carcinogenesis of liver cells.
A l t h o u g h regulators of proliferation h a v e b e e n identified for the regenerative liver, the m o l e c u l a r control of cellular proliferation a n d differentiation in the d e v e l o p i n g liver r e m a i n s largely undefined. W e h a v e p r e v i o u s l y s h o w n that the d e v e l o p i n g liver regenerates i n d e p e n d e n t l y of interleukin-6 following a neonatal-onset liver injury. Based on these data, w e h y p o t h e s i z e d that transcriptional r e g u l a t i o n for cellular hyperplasia a n d differentiation is n o t shared bet w e e n physiologic a n d regenerative growth. To address this hypothesis, w e d e t e r m i n e d the global gene expression of m u r i n e livers d u r i n g two phases of physiologic g r o w t h ( e m b r y o n i c day 14.5 p.c. a n d postnatal day 14) a n d initial phases o f liver r e g e n e r a t i o n (6 a n d 24 h o u r s after 70% partial h e p a t e c t o m y ) ; livers f r o m u n c h a l l e n g e d a n d s h a m - o p e r a t e d adult m i c e w e r e also i n c l u d e d as controls. Hepatic m R N A was c o n v e r t e d to Cy3-Cy5 c D N A pools, a n d each s a m p l e w a s h y b r i d i z e d to the M o u s e GEM1 m i c r o a r r a y s f r o m Incyte G e n o m i c s c o n t a i n i n g 8,734 gene products. M i n i n g of all transcripts s i m u l t a n e o u s l y identified 63 gene p r o d u c t s o v e r e x p r e s s e d 2-fold or h i g h e r in one or m o r e g r o w t h states. Cluster analysis s h o w e d segregation of these genes into each experim e n t a l g r o u p , w i t h m i n i m a l overlap in the transcriptional profile b e t w e e n physiologic a n d regenerative growth. Analysis of each g r o u p revealed signature genes w h i c h s h a r e d similar function. For d e v e l o p i n g livers at e m b r y o n i c day 14.5, w e f o u n d a p r e d o m i n a n t o v e r e x p r e s s i o n of genes related to h e m a topiesis, while livers at postnatal day 14 displayed transcripts for metabolic regulators. Notably, genes regulating i n f l a m m a t i o n a n d m e t a b o l i s m w e r e highly expressed in regenerative livers. Only 7 genes s h a r e d overexpression at b o t h postnatal day 14 a n d liver regeneration, a n d out of these genes, 4 are i n v o l v e d in m e t a b o l i s m a n d inflammation. Multi-tissue s u r v e y of genes highly expressed in postnatal and/or regenerative livers across 76 n o r m a l a n d experi m e n t a l m u r i n e tissues identified an u n i q u e temporo-spatial p a t t e r n of expression restricted to the liver for the n o v e l i n f l a m m a t i o n - r e l a t e d genes CD14, o r o s o m u c o i d 1, hepcidin, serine protease inhibitor 2.1, Ith3, a n d for the m i tochondrial translocator T i m 4 4 . I n conclusion: 1) hepatic gene expression d u r i n g pre- a n d post-natal d e v e l o p m e n t reflects the g o v e r n i n g function of h e m a t o p o i e s i s d u r i n g e m b r y o g e n e s i s a n d acquisition of differentiated m e t a bolic function in postnatal livers, a n d 2) liver gene expression d u r i n g postnatal d e v e l o p m e n t does n o t fully recapitulate the transcriptional profile of the regenerative liver, w h i c h s h o w s overexpression for only a small n u m b e r of genes related to g r o w t h a n d m e t a b o l i s m . T h e s e data u n d e r s c o r e the n e e d of the developing liver to acquire specialized metabolic function, while it m a i n t a i n s cellular proliferation to allow for physiologic growth.
AASLD ABSTRACTS
HEPATOLOGY O c t o b e r 2 0 0 1
847
848
RECEPTOR TARGETING TO HEPATOCYTES INCREASES POLYETHYLE N I M I N E - M E D I A T E D NONVIRAL DELIVERY OF NUCLEIC ACIDS TO THE LIVER. Paul D Richardson, Rod Felsheim, Gladys Chuey, Betsy T Kren, Clifford J Steer, U n i v e r s i t y of Minnesota Medical School, Minneapolis, M N
O S T E O A C T M N , A GENE ISOLATED FROM THE LIVER OF RAT FED W I T H A CHOLINE-DEFICIENT, L-AMINO ACID DEFINED DIET, ACCELERATED INVASION AND METASTASIS OF tIEPATOMA CELLS. Masaaki Onaga, Akio Ido, Akihiro Moriuti, Yukiko O n a g a , Satoru Hasnike, H i r o f u m i Uto, Kenji Nagata, Takeshi Hori, Shuichi Hirono, Katsuhiro Hayashi, Hirohito T s u b o u c h i , Miyazaki Medical College, Kiyotake T o w n J a p a n
There are now well-defined fundamental aspects of any successful gene therapy/targeted gene repair process. These include efficient packaging of DNA; effective delivery and cell specificity with minimal toxicity-; and efficient nuclear translocation. The liver is an attractive organ for gene therapy because it is central to many inherited and acquired genetic disorders. In addition, a large blood supply enables efficient delivery and extravasation of complexes through fenestrae. The asialoglycoprotein receptor is unique to hepatocytes and highly expressed on the cell surface membrane. Polyethylenimine (PEI) is a cationic polymer able to condense DNA and afford adequateprotection against nuelsases. PEI possesses a large number of uncharged amines that enable it to act as a proton sponge after cellular uptake, preventin~ vesicle acidification, inhibiting intracellular nucleases and allowing osmotic rupture ot the endosomes. In addition, PEI may also possess nuclear localizing properties. Unlike some other polycations, PEI is an effective agent in serum, which is obviously critical for in vivo delivery. Lactosylation of the PEI enables targeting to the asialoglycoprotein receptor and hence mediates hepatocyte specificity. The AIM of these studies was to optimize the delivery of both the iuciferase reporter plasmid pGL3 and florescently-labeled single-stranded oligonucleotides (SSOs) to human hepatoma HUH-7 cells, primary rat hepatocytes and primary mouse fibroblasts. We have recently shown that SSOs are effective mediators of site-specific gene repair when delivered in high copy number to cells. METHODS: Branched 10 kDa and linear and branched 25 kDa formulations of PEI were used. Lactose was covalentiy attached to either the primary or secondary amines on the branched PEIs by EDAC conjugation or Schiff-base reaction with titanium isopropoxide, respectively. The efficacy of delivery for each of these different formulations was compared using a reporter plasmid encoding lucfferase and a Cy-3-1abeled 35 mer SSO. The relative sizes of the PEI:DNA complexes were examined by transmission electron microscopy (TEM), and nuclear delivery assessed by confocal microscopy. Luciferase activity was determined by a luminometric assay. RESULTS: Delivery of the plasmid was most efficient with the branched 25 kDa PEI resulting in luciferase activity > 10 t° RLU/mg protein. This was an order of magnitude greater than the branched 10 kDa or linear 25 kDa PEI, but was generally more toxic. Lactosylation of the PEI resulted in a marked increase in uptake by hepatocytes, which was competitively blocked by coincubation with EGTA or D-galactose. In contrast, no significant changes in uptake were Observed with fibroblasts. Primary amine lactosylation increased the delivery efficiency by at least 2 orders of magnitude over secondary amine conjugation. Linear PEI was most effective in delivering SSOs and resulted in a more rapid and significantly increased nuclear localization relative to the branched PEIs. In fact, there was marked nuclear uptake within 1 hour of transfection. Under all conditions, delivery was significantly reduced in primary rat hepatocytes compared to HUH-7 cells. TEM of the 25 kDa 7 kb plasmid complexes were < 50 nm in size and monodisperse. Interestingly, the branched 10/25 kDa combination exhibited larger complexes of ~ 65 nm. The linear complexes tended to aggregate and were typically > 100 nm in size. CONCLUSIONS: PEI is an effective agent in delivering both plasmids and small nucleic acids to hepatocytes and primary fibroblasts. The optimal formulation of PEI was clearly influenced by the relative size of the nucleic acid molecule delivered to the cell. Lactosylation of PEI provides a simple modification to significantly improve specificity and uptake by hepatocytes. The PEI polycatinn remains a versatile and potentially effective agent in the delivery of nucleic acids for gene therapy.
BACKGROUND: The development of hepatocellular carcinoma (HCC) is associated with chronic liver disease, particularly cirrhosis. However, little is known about the genes involved in hepatocarcinogenesis associated with cirrhosis. In a rat model of hepatocarcinogenesis induced by a choline-deficient, L-amino acid defined (CDAA) diet, initial steatosis and subsequent cirrhosis is followed by occurrence of HCC. In this study, to clarify the genes associated with hepatocarcinogenesis at an early phase, we examined the genes expressed in the liver of rats fed a CDAA diet for 2 months and isolated osteoactivin (OA) cDNA. We also analyzed the OA expression in human HCC tissues and the effect of OA on invasion and metastasis of hepatoma cells. Materials and Methods: (1) The genes expressed in the liver of rats fed with a CDAA diet for 2 months were isolated using suppression subtractive hybridization. (2) Northern blot analysis was performed to examine the expression of OA mRNA in the liver of rats fed with a CDAA diet, human HCC and normal liver tissues. (3) OA- or aatisense-OA cDNA was introduced into rat hepatoma cells (dRLh84), and G418-resistant clorlal cell lines (dRLh84/OA or dRLh84/AS-OA, respec~ tively) were established. (4) The effect of OA on invasiveness in the presence or absence of hepatocyte growth factor (HGF) was examined by Matrigel invasion assays. (5) To analyze the OA-mediated metastasis, dRLH84/parent and dRLH84/OA were inoculated into the spleen of nude mice, and the development of hepatic metastasis was evaluated. RESULTS: (1) We isolated transferrin, NF-KB, uncoupling protein-2 and OA cDNAs. (2) OA mRNA was strongly expressed in the liver of rat fed with a CDAA diet for 1 to 3 months and moderate expression continued until 18 months. Also, in the HCC developing in the rat given a CDAA diet for 18 months, a higher level of OA mRNA expression was observed. In humans, OA mRNA expression was detected in both HCC and the tissues surrounding the lesion, whereas OA was not expressed in normal liver. The level of OA expression in well-differentiated HCCs was higher than that in the surrounding liver tissues as well as moderately- and poorly-differentiated HCCs. (3) The expression of OA mRNA increased twice in dRLh84/OA cells and decreased to 30% in dRLh84/AS-OA ceils, as compared with dRLh84/parent. (4) The number ofdRLh84/OA ceils invading the Matrigel membrane was 10-tbld higher than that of dRLh84/parem (p<0.05). HGF stimulated the invasiveness of dRLh84/parental cells by 50-fold. However, HGF treatment induced only a 4-fold increase in the Matrigel invasiveness of dRLh84/AS-OA ceils (p<0.001). (5) When dRLh84/parental cells were inoculated into the spleen of nude mice, metastatic liver tumors were observed in 12.5% (1/8) of mice. In contrast, intrasplenic inoculation of dRLh84/OA cells resulted in hepatic metastasis in all mice (16/16) (p<0.005). ICONCLUSIONS] We isolated OA gene from the rat liver during the early phase of a CDAA diet-induced hepatocarcinogenesis. OA was strongly expressed in not only rat HCC tissues but also human HCC tissues, and OA overexpression accerelated the invasiveness and metastasis of hepatoma ceils, These results suggest that OA is a novel factor involved in hepatocarcinogenesis and tumor progression through the stimulation of tumor invasiveness and metastasis of HCC.
849
850
FUNCTIONAL COOPERATION OF P18 INK4c W I T H P21 cwl/wAF1 AND P27 ~P1 IN THE REGENERATING LIVER. T o m Luedde, D e p a r t m e n t of Gast r o e n t e r o l o g y a n d H e p a t o l o g y , Medizinische H o c h s c h u l e H a n n o v e r , Germ a n y , H a n n o v e r G e r m a n y ; Maria E Rodriguez, L i n e b e r g e r C o m p r e h e n s i v e C a n c e r Center, U N C at Chapel Hill, NC, Chapel Hill, NC; Christian T r a u t w e i n , D e p a r t m e n t of G a s t r o e n t e r o l o g y a n d Hepatology, Medizinische H o c h s c h u l e H a n n o v e r , G e r m a n y , H a n n o v e r G e r m a n y ; David A Brenner, Dep a r t m e n t of Digestive Diseases a n d Nutrition, U N C at Chapel Hill, NC, Chapel Hill, NC; Yue Xiong, L i n e b e r g e r C o m p r e h e n s i v e C a n c e r Center, U N C at Chapel Hill, NC, C h a p e l Hill, N C
DEVELOPING LIVERS DISPLAY AN UNIQUE TRANSCRIPTIONAL PROGRAM W I T H MINIMAL OVERLAP W I T H HEPATIC REGENERATION. N a n c y Kelley-Loughnane, G r e g g Sabla, Bruce J A r o n o w , Jorge A Bezerra, Children's Hospital Medical Center, Cincinnati, O H
Entry of quiescent cells into the cell cycle is driven by the cyclin D-dependent kinases Cdk4 and Cdk6. pl8 INK4eis a member of the INK4 family of proteins that specifically target the Cdk4 and Cdk6 kinases and inhibit their catalytic activity. The ability of p 18 to control G1 phase progression has been studied in vitro, but less is known about the significance of this inhibition in vivo. As such, the role of p18 for cell cycle control in the liver is unclear. In this study, we used mice lacking p18zNK~c,p 2 t cwzAvAFl or p27KIP1alone and mice lacking both p18/p21 or p18/p27. The mice were analyzed for differences in the timing of ceil cycle progression after partial hepatectomy (PH). An IP-Western showed that in wild type (WT) mice p18 is expressed at a lower level in the quiescent liver compared to NIH 3T3 cells. After PH this basic level remained unchanged during the first 96h. In the p18 deficient mice PCNA expression as a marker for S-Phase entry and Cyclin D expression revealed no changes compared to the WT control mice with maximum expression at 48h after PH. BrdU-staining used as an additional marker for S-Phase entry of ceils showed that, like in the control group, the majority of nuclei (~50%) were positive 48h after PH and almost no positive nucleus was found after 36h. Loss of p21 function resuhed in a shorter G1 interval: Both PCNA and cycfin D1 were expressed at 36h post-PH with maximum expression at 48h post-PH. At 36h after PH about 10% of the nuclei stained positive for BrdU, but the majority of nuclei (~40%) were positive after 48h. The group of p27 mutant mice had a minimally increased level of PCNA expression prior to PH compared to WT control. However, upregulation of PCNA expression to S-Phase specific levels occurred no earlier than 48h after PH. Cyelin D expression and BrdU staining showed no changes to the WT control group. In the mice lacking both pl8 and p21 we found that the G1 Phase interval after PH was even shorter than in the p21 single knockout animals. PCNA as well as Cyclin D expression began already 24h after PH with maximum expression at 36h after PH. BrdU staining showed that the majority of nuclei (~40%) were already positive 36h post PH and only"a minor portion was still positive after 48h (~10%). Finally the plB/p27 double-knockout mice showed an even stronger increase of base level PCNA expression and a stronger maximum compared to both the WT control animals and p27 null-mice, but no changes in the timing of the expression was evident. Cyclin D expression remained unchanged in the p18/p27 double mutant animals compared to both groups. Furthermore at 36h post-PH almost no nuclei were BrdU-positive whereas 48h post-PH almost 90% of hepatocytes were BrdU-positive. Our results demonstrate for the first time that pl8, although expressed at a low level in the liver, is important for controlling cell cycle progression in proliferating hepatocytes in vivo. Moreover, our study indicates that pig collaborates with other inhibitors of cyclin dependent kinases to regulate different aspects of cell cycle progression: p 18 and p21 collaborate in regulating the length of the G1 phase after PH while pi8 and p27 seem to cooperate in modulating the threshold of bepatocytes to enter the cell cycle as disruption of these two genes leads to almost double the number of hepatocytes that synchronously enter the cell cycle after PH. These findings could also have implications for the carcinogenesis of liver cells.
A l t h o u g h regulators of proliferation h a v e b e e n identified for the regenerative liver, the m o l e c u l a r control of cellular proliferation a n d differentiation in the d e v e l o p i n g liver r e m a i n s largely undefined. W e h a v e p r e v i o u s l y s h o w n that the d e v e l o p i n g liver regenerates i n d e p e n d e n t l y of interleukin-6 following a neonatal-onset liver injury. Based on these data, w e h y p o t h e s i z e d that transcriptional r e g u l a t i o n for cellular hyperplasia a n d differentiation is n o t shared bet w e e n physiologic a n d regenerative growth. To address this hypothesis, w e d e t e r m i n e d the global gene expression of m u r i n e livers d u r i n g two phases of physiologic g r o w t h ( e m b r y o n i c day 14.5 p.c. a n d postnatal day 14) a n d initial phases o f liver r e g e n e r a t i o n (6 a n d 24 h o u r s after 70% partial h e p a t e c t o m y ) ; livers f r o m u n c h a l l e n g e d a n d s h a m - o p e r a t e d adult m i c e w e r e also i n c l u d e d as controls. Hepatic m R N A was c o n v e r t e d to Cy3-Cy5 c D N A pools, a n d each s a m p l e w a s h y b r i d i z e d to the M o u s e GEM1 m i c r o a r r a y s f r o m Incyte G e n o m i c s c o n t a i n i n g 8,734 gene products. M i n i n g of all transcripts s i m u l t a n e o u s l y identified 63 gene p r o d u c t s o v e r e x p r e s s e d 2-fold or h i g h e r in one or m o r e g r o w t h states. Cluster analysis s h o w e d segregation of these genes into each experim e n t a l g r o u p , w i t h m i n i m a l overlap in the transcriptional profile b e t w e e n physiologic a n d regenerative growth. Analysis of each g r o u p revealed signature genes w h i c h s h a r e d similar function. For d e v e l o p i n g livers at e m b r y o n i c day 14.5, w e f o u n d a p r e d o m i n a n t o v e r e x p r e s s i o n of genes related to h e m a topiesis, while livers at postnatal day 14 displayed transcripts for metabolic regulators. Notably, genes regulating i n f l a m m a t i o n a n d m e t a b o l i s m w e r e highly expressed in regenerative livers. Only 7 genes s h a r e d overexpression at b o t h postnatal day 14 a n d liver regeneration, a n d out of these genes, 4 are i n v o l v e d in m e t a b o l i s m a n d inflammation. Multi-tissue s u r v e y of genes highly expressed in postnatal and/or regenerative livers across 76 n o r m a l a n d experi m e n t a l m u r i n e tissues identified an u n i q u e temporo-spatial p a t t e r n of expression restricted to the liver for the n o v e l i n f l a m m a t i o n - r e l a t e d genes CD14, o r o s o m u c o i d 1, hepcidin, serine protease inhibitor 2.1, Ith3, a n d for the m i tochondrial translocator T i m 4 4 . I n conclusion: 1) hepatic gene expression d u r i n g pre- a n d post-natal d e v e l o p m e n t reflects the g o v e r n i n g function of h e m a t o p o i e s i s d u r i n g e m b r y o g e n e s i s a n d acquisition of differentiated m e t a bolic function in postnatal livers, a n d 2) liver gene expression d u r i n g postnatal d e v e l o p m e n t does n o t fully recapitulate the transcriptional profile of the regenerative liver, w h i c h s h o w s overexpression for only a small n u m b e r of genes related to g r o w t h a n d m e t a b o l i s m . T h e s e data u n d e r s c o r e the n e e d of the developing liver to acquire specialized metabolic function, while it m a i n t a i n s cellular proliferation to allow for physiologic growth.