- Email: [email protected]
Gene therapy of hepatocellular carcinoma using the α-fetoprotein gene in an adenoviral vector
158A
205
20"/
AASLD A B S T R A C T S
TRANSGENIC MICE EXPRESSING A Zn-INDUCIBLE DOMINANT N E G A T I V E T Y P E II T G F f l R E C E P T O R : A M O D E L FOR A B R O G A T I O N O F T H E ACTIONS O F TGFB IN LIVER. RA Serra, S Sitaric!, HL Moses. W E Russell. The Departments of Cell Biology and Pediatrics, Vanderbilt University, Nashville, TN 37232. TGFI~ is a potent inhibitor of hepatocyte proliferation and has been proposed to be the major negative growth regulator in regenerating liver after partial hepatectomy. TGFB signals through a heteromeric complex comprised of Type I and Type II serine/threonine ldnases. To evaluate the role of TGF/~ in the control of liver growth, regeneration, and carcinogenesis, we have produced transgenic mice (MTDNIIR-Iine 15; MTR-15) that express a kinase defective, dominant-negative TGFB type II receptor under control of a metallothionein promoter. The dominant negative receptor has been shown to block the effects of TGFB when expressed in MvILu cells in culture, and the metallothionein promoter allows expression of the mutant receptor to be regulated with zinc. Transgene expression was induced in primary mouse hepatocytes cultured in the presence of 100200#M ZnS04. In the absence of Zn, transgene expression was low to undetectable, and MTR-15 hepatocytes Showed normal sensitivity to the growth inhibitory actions of TGFg on EGF-stimulated DNA synthesis (kI~20pM). In the presence of 100 # M Zn, however, concentrations of TGFI~ as high as 200 pM failed to be inhibitory. Transgene expression was maximally induced in the livers of MTR-15 mice within 3h of a single i~p. dose of ZnC12, 10 mg/kg. These studies suggest the utility of the MTR-15 mouse as a potent tool to study the hepatic actions of TGFB, both in vitro and in vivo. This model should permit studies on the molecular mechanisms of TGFI~ action in transgene-expressing hepatic cells, and a means to dissect both the role and temporal frame of action of TGF~ in the control of liver development, regeneration, fibrosis, and carcinogenesis.
206
THE LIVER EDGE AND THE SUBCAPSUIAR IAYER ARE DIFFERENT FROM THE DEEPER PART OF THE LIVER LOBE: LOCAL HETEROGENEITY OF THE "SHELL"AND THE ~CORE" IN MAMMALIAN LIVERS.
208
W Ekataksin.WNEkataksin,CC Ekataksin.W Hirunsi,and KWake. Departmentof Anatomy, Schoolof Medicine,TokyoMedicaland DentalUniversity,Tokyo,Japan. Although manypathologistssuspect the inhomogeneityof liversubstance, the idea that • the organ is significantlyheterogeneousdependingon its locationwithinthe lobe has so far not beenconvincinglycorroborated. Westudiedontogeniclythe lobularionand vascularization in pig fetusesand at the age of 1, 2, 7, and 17 days, 3 weeks, 6 months, 1.5 and 3 years,and confirmedsome of the findingsin rat by lightand scanningelectron microscopy(injectioncorrosion casts). Lobularionwas not manifestedin fetusesand earlyneonates, but became discerniblewithtrace demarcationin the deeper liverat day 17. Fromweek 3 on, the typical fibrousseptationspreadfromhilumthroughoutthe lobesdistinguishingtwo lobuletypes,the simple hepatic lobule~ (SILL),isolableunitswithoutingrownsepta,and the compound hepatic lobules ((:HI.), partlypenetratedby innaiobularsepta incompletelysplittingthe lobules. At week 3 most lobules,81%, were CHL,being present more, 93%, in the subcapsularlayer. Lobulesgrewin SHLproportion,decreasingCHLproportion,withadvancingage,and enlarged in size. Smalllobuleswere predominantsabcapsularly,especiallyat the liveredge; giant lobules,5000-8000k~mlong,usuallyoccurredborderinglargevesselsin the depth. Tracing 3292 lobulesfrom serialsectionsat 3 weeks,6 months, 1.5 and 3 yearsrevealedthat in the subcapsularlayer,SHLincreasedas 7, 13, 20, and 48%,whereasin the deeper subpopulation, these were 24, 28, 53, and 57%, respectively. Dividinglobules were frequentlyseen subperitoneally,perpendicularlobulesbeinghorizontallyhaffed. The septalbranchesof the portal vein developed and elongatedwith the lobule growth. The axialcentral venules extendedwith the expansionof the lobules,highlydendriticin the CIlL. The hepaticartery latelygrewinto the fetallivers,barelyseen beyondlargeportaltracts,at crown-romplengthof 4 and 7 cm. Capsulararterioles,30-60 p.m diameter,were rare at day 1, 2, and 7, but became frequent at day 17 and week3, and dense in adult. In rat, arterialvascularbeds were limited to the deeper part, being sparse near surface of liversof neonate, whereas in adult, rich arterial network permeated most superficialportal tracts; capsulararteriolewas occasional. Findings are suggestiveof the locality, a third level of heterogeneitybased on different locations,addingto the zonality and the regionality. The "shell"and the "core"of the liver lobes havedifferentgrowthprofileswith differinglobulecompositionand vasculature. SHL representa well-vascularizedphenotype,beingfoundlessinyoungeranimals. The subcapsular subpopulationforma "hepatogenic"layer,more or lessanalogousto the corticalnephrogenic zone in the kidney. (Supportedin part by Grant-in.Aid#07770006).
GENE
HEPATOLOGY October 1995 THERAPY
OF
HEPATOCELLULAR
CARCINOMA
U S I N G T H E a - F E T O P R O T E I N G E N E IN AN A D E N O V I R A L V E C T O R . S Kanek0*§~E Matsushita*§, Y Chiang#, P HaUenbeck#, T _Kotani#, T Tamaokff, H NakabavashiL K Kobavashi§, WF Anderson*. *i University of Southern California; #. Genetic Therapy Inc:, USA; ~, University of Calgary, Canada; §. 1st Dept. of Int. Med., Kanazawa University, Kanazawa, Japan. The majority of patients with hepatoeallular carcinoma have an elevated a-fetoprotein (AIP).level. Using the 5'-flanking sequence as a promoter for the herpes simplex virus thymidine kinase (HSV-TK) gene in an adenoviral vector (AvlAFPTK1), the therapeutic efficacy of adenoviral mediated HSV-TK transduction followed by ganciclovir (GCV) administration was studied in tumors in athymic nude mice. AvlAFFFK1 transduction of two cell lines demonstrated HSV:TK enzyme activity only in the AFP producing cells (HUH7, 1757cpm at MOI 5000) and not in the AFP non-producing cells (SK-Hep-1, 43cpm at MOI 5000). As expected, only transduced HuH7 cells were killed by GCV treatment. Transduetion by an adenoviral vector harboring a Rous sarcoma virus promoter and HSV-TK gene (Av 1TK1) showed enzymatic activity and GCV killing in both c;ell lines (Huh7, 17472cpm at MOI !00; SK-Hep1, 5270cpm at MOI 100). All HuH7 tumors which were transduced with either AvlAFPTK1 (n=5) or AvlTK1 (n=5), completely regressed after GCV treatment. On the other hand, there was ,complete regression of SKHep-1 tumors only when treated with Av 1TK1 and GCV (n=5), and not when treated with AvlAFPTK1 and GCV (n=5). Thus, cell specific killing was achieved by adenoviral vector containing AFP promoter fo~"the HSV-TK gene and GCV treatment. Adenoviral mediated gene therapy using the HSV-TK/GCV system with the AFP gene appears to have the potential for the treatment of human hepatocellular carcinoma.
PROGNOSTIC VALUE OF THE PERFUSED HEPATIC MA.~% JC Hoefs. H Iloefs~ F Wane. G Kanel. Liver Disease Program, Dept. of Medicine, Division of Gastroenterology, Dept. of Radiology, Division of Nuclear Medicine, University of California, Irvine and Dept. of Pathology, Division of Liver Unit Laboratories, Univ. of Southern California and la)s Rancho Amigos Hospital, Downey, California. Redistribution uf Tc~ Sulfur Colloid as measured on the SPECT liver spleen scan (LSS) is thought to measure the perfused hepatic mass (PilM) because of the correlation with the functional hepatic mass and liver disease severity. Ilowever, only a few small studies have asse~ssed the prognostic value of the PHM as measured by LSS. The survival a t 1, 2, and 3 years was assessed in 258 patients with the histnlogic equivalent of marked fibrosis ur cirrhosis who alsa had a L~;S near the time of histologic assessment. The effect Of liver transplant was included. METHODS: 258 patients with histolugic cirrhosis were followed to death or at least 1 year of follow-up from the tlme of LSS, LSS distribution of sulfur colh)id was expressed from SPECT L~;S as a liver bone marrow index and liver spleen index. The perfused hepatic mass (PHM) is the mean of these twn indices (normal > 85) and was divided into 10 unit ranges of PHM fur survival assessment. RESULTS: The overall cumulative survival in these patients was 76% (258 patients) at 1 year, 62% (224 patients) at 2 years and $6.6% (200 patients) at 3 years and include 27 patients wire were transplanted. The overall survival in the PMIl unit ranges are shown in the table: % SURVIVAL PER PHM RANGE YEAR <40 4q-49 50-59 60-69 70-79 80-89 90-99 >100
1
833 92.1 92.9 81.1 69.4 86.1 82.9 75.9 3 53.9 79.3 67.9 75.9 Deaths were due to liver disease (61), Hepatocellular Carcinoma (8), post-liver transplant (9) and from ram-hepatic c~mses (16), inclnding AIDS (5). The percent of deaths due to CLII or IlCC in the first year decreased frnm 90-11~% beh)w a PIlM of 60 to 75-85% between 60-8(l to 28-30% above 80. In year 2 and 3, 90% of all deaths were due to CLD nr IlCC with a PHM < 80. The correlation between the mid-range PHM from 35 to 85 and 1 year survival was highly significant (P<.00I): % survival = 1.4637 PHM - 29; r=.99. The survival without death or transplant in patients without HCC was correlated (P < .(J01) with the PMH at 1 year (% survival = 1.18 PHM-29) and 3 years (% survival = 1.25 PHM-391. CONCLUSIONS: 11 The PHM measured by LSS was closely correlated with prognosis and 2) patients wire died with a normal PHM generally died of non-CLD related problems. 2
23.1 9.1 9.1
54.2 59.3 33.3 10.0 33.3 29.2
743 61.8 57.1
205
20"/
AASLD A B S T R A C T S
TRANSGENIC MICE EXPRESSING A Zn-INDUCIBLE DOMINANT N E G A T I V E T Y P E II T G F f l R E C E P T O R : A M O D E L FOR A B R O G A T I O N O F T H E ACTIONS O F TGFB IN LIVER. RA Serra, S Sitaric!, HL Moses. W E Russell. The Departments of Cell Biology and Pediatrics, Vanderbilt University, Nashville, TN 37232. TGFI~ is a potent inhibitor of hepatocyte proliferation and has been proposed to be the major negative growth regulator in regenerating liver after partial hepatectomy. TGFB signals through a heteromeric complex comprised of Type I and Type II serine/threonine ldnases. To evaluate the role of TGF/~ in the control of liver growth, regeneration, and carcinogenesis, we have produced transgenic mice (MTDNIIR-Iine 15; MTR-15) that express a kinase defective, dominant-negative TGFB type II receptor under control of a metallothionein promoter. The dominant negative receptor has been shown to block the effects of TGFB when expressed in MvILu cells in culture, and the metallothionein promoter allows expression of the mutant receptor to be regulated with zinc. Transgene expression was induced in primary mouse hepatocytes cultured in the presence of 100200#M ZnS04. In the absence of Zn, transgene expression was low to undetectable, and MTR-15 hepatocytes Showed normal sensitivity to the growth inhibitory actions of TGFg on EGF-stimulated DNA synthesis (kI~20pM). In the presence of 100 # M Zn, however, concentrations of TGFI~ as high as 200 pM failed to be inhibitory. Transgene expression was maximally induced in the livers of MTR-15 mice within 3h of a single i~p. dose of ZnC12, 10 mg/kg. These studies suggest the utility of the MTR-15 mouse as a potent tool to study the hepatic actions of TGFB, both in vitro and in vivo. This model should permit studies on the molecular mechanisms of TGFI~ action in transgene-expressing hepatic cells, and a means to dissect both the role and temporal frame of action of TGF~ in the control of liver development, regeneration, fibrosis, and carcinogenesis.
206
THE LIVER EDGE AND THE SUBCAPSUIAR IAYER ARE DIFFERENT FROM THE DEEPER PART OF THE LIVER LOBE: LOCAL HETEROGENEITY OF THE "SHELL"AND THE ~CORE" IN MAMMALIAN LIVERS.
208
W Ekataksin.WNEkataksin,CC Ekataksin.W Hirunsi,and KWake. Departmentof Anatomy, Schoolof Medicine,TokyoMedicaland DentalUniversity,Tokyo,Japan. Although manypathologistssuspect the inhomogeneityof liversubstance, the idea that • the organ is significantlyheterogeneousdependingon its locationwithinthe lobe has so far not beenconvincinglycorroborated. Westudiedontogeniclythe lobularionand vascularization in pig fetusesand at the age of 1, 2, 7, and 17 days, 3 weeks, 6 months, 1.5 and 3 years,and confirmedsome of the findingsin rat by lightand scanningelectron microscopy(injectioncorrosion casts). Lobularionwas not manifestedin fetusesand earlyneonates, but became discerniblewithtrace demarcationin the deeper liverat day 17. Fromweek 3 on, the typical fibrousseptationspreadfromhilumthroughoutthe lobesdistinguishingtwo lobuletypes,the simple hepatic lobule~ (SILL),isolableunitswithoutingrownsepta,and the compound hepatic lobules ((:HI.), partlypenetratedby innaiobularsepta incompletelysplittingthe lobules. At week 3 most lobules,81%, were CHL,being present more, 93%, in the subcapsularlayer. Lobulesgrewin SHLproportion,decreasingCHLproportion,withadvancingage,and enlarged in size. Smalllobuleswere predominantsabcapsularly,especiallyat the liveredge; giant lobules,5000-8000k~mlong,usuallyoccurredborderinglargevesselsin the depth. Tracing 3292 lobulesfrom serialsectionsat 3 weeks,6 months, 1.5 and 3 yearsrevealedthat in the subcapsularlayer,SHLincreasedas 7, 13, 20, and 48%,whereasin the deeper subpopulation, these were 24, 28, 53, and 57%, respectively. Dividinglobules were frequentlyseen subperitoneally,perpendicularlobulesbeinghorizontallyhaffed. The septalbranchesof the portal vein developed and elongatedwith the lobule growth. The axialcentral venules extendedwith the expansionof the lobules,highlydendriticin the CIlL. The hepaticartery latelygrewinto the fetallivers,barelyseen beyondlargeportaltracts,at crown-romplengthof 4 and 7 cm. Capsulararterioles,30-60 p.m diameter,were rare at day 1, 2, and 7, but became frequent at day 17 and week3, and dense in adult. In rat, arterialvascularbeds were limited to the deeper part, being sparse near surface of liversof neonate, whereas in adult, rich arterial network permeated most superficialportal tracts; capsulararteriolewas occasional. Findings are suggestiveof the locality, a third level of heterogeneitybased on different locations,addingto the zonality and the regionality. The "shell"and the "core"of the liver lobes havedifferentgrowthprofileswith differinglobulecompositionand vasculature. SHL representa well-vascularizedphenotype,beingfoundlessinyoungeranimals. The subcapsular subpopulationforma "hepatogenic"layer,more or lessanalogousto the corticalnephrogenic zone in the kidney. (Supportedin part by Grant-in.Aid#07770006).
GENE
HEPATOLOGY October 1995 THERAPY
OF
HEPATOCELLULAR
CARCINOMA
U S I N G T H E a - F E T O P R O T E I N G E N E IN AN A D E N O V I R A L V E C T O R . S Kanek0*§~E Matsushita*§, Y Chiang#, P HaUenbeck#, T _Kotani#, T Tamaokff, H NakabavashiL K Kobavashi§, WF Anderson*. *i University of Southern California; #. Genetic Therapy Inc:, USA; ~, University of Calgary, Canada; §. 1st Dept. of Int. Med., Kanazawa University, Kanazawa, Japan. The majority of patients with hepatoeallular carcinoma have an elevated a-fetoprotein (AIP).level. Using the 5'-flanking sequence as a promoter for the herpes simplex virus thymidine kinase (HSV-TK) gene in an adenoviral vector (AvlAFPTK1), the therapeutic efficacy of adenoviral mediated HSV-TK transduction followed by ganciclovir (GCV) administration was studied in tumors in athymic nude mice. AvlAFFFK1 transduction of two cell lines demonstrated HSV:TK enzyme activity only in the AFP producing cells (HUH7, 1757cpm at MOI 5000) and not in the AFP non-producing cells (SK-Hep-1, 43cpm at MOI 5000). As expected, only transduced HuH7 cells were killed by GCV treatment. Transduetion by an adenoviral vector harboring a Rous sarcoma virus promoter and HSV-TK gene (Av 1TK1) showed enzymatic activity and GCV killing in both c;ell lines (Huh7, 17472cpm at MOI !00; SK-Hep1, 5270cpm at MOI 100). All HuH7 tumors which were transduced with either AvlAFPTK1 (n=5) or AvlTK1 (n=5), completely regressed after GCV treatment. On the other hand, there was ,complete regression of SKHep-1 tumors only when treated with Av 1TK1 and GCV (n=5), and not when treated with AvlAFPTK1 and GCV (n=5). Thus, cell specific killing was achieved by adenoviral vector containing AFP promoter fo~"the HSV-TK gene and GCV treatment. Adenoviral mediated gene therapy using the HSV-TK/GCV system with the AFP gene appears to have the potential for the treatment of human hepatocellular carcinoma.
PROGNOSTIC VALUE OF THE PERFUSED HEPATIC MA.~% JC Hoefs. H Iloefs~ F Wane. G Kanel. Liver Disease Program, Dept. of Medicine, Division of Gastroenterology, Dept. of Radiology, Division of Nuclear Medicine, University of California, Irvine and Dept. of Pathology, Division of Liver Unit Laboratories, Univ. of Southern California and la)s Rancho Amigos Hospital, Downey, California. Redistribution uf Tc~ Sulfur Colloid as measured on the SPECT liver spleen scan (LSS) is thought to measure the perfused hepatic mass (PilM) because of the correlation with the functional hepatic mass and liver disease severity. Ilowever, only a few small studies have asse~ssed the prognostic value of the PHM as measured by LSS. The survival a t 1, 2, and 3 years was assessed in 258 patients with the histnlogic equivalent of marked fibrosis ur cirrhosis who alsa had a L~;S near the time of histologic assessment. The effect Of liver transplant was included. METHODS: 258 patients with histolugic cirrhosis were followed to death or at least 1 year of follow-up from the tlme of LSS, LSS distribution of sulfur colh)id was expressed from SPECT L~;S as a liver bone marrow index and liver spleen index. The perfused hepatic mass (PHM) is the mean of these twn indices (normal > 85) and was divided into 10 unit ranges of PHM fur survival assessment. RESULTS: The overall cumulative survival in these patients was 76% (258 patients) at 1 year, 62% (224 patients) at 2 years and $6.6% (200 patients) at 3 years and include 27 patients wire were transplanted. The overall survival in the PMIl unit ranges are shown in the table: % SURVIVAL PER PHM RANGE YEAR <40 4q-49 50-59 60-69 70-79 80-89 90-99 >100
1
833 92.1 92.9 81.1 69.4 86.1 82.9 75.9 3 53.9 79.3 67.9 75.9 Deaths were due to liver disease (61), Hepatocellular Carcinoma (8), post-liver transplant (9) and from ram-hepatic c~mses (16), inclnding AIDS (5). The percent of deaths due to CLII or IlCC in the first year decreased frnm 90-11~% beh)w a PIlM of 60 to 75-85% between 60-8(l to 28-30% above 80. In year 2 and 3, 90% of all deaths were due to CLD nr IlCC with a PHM < 80. The correlation between the mid-range PHM from 35 to 85 and 1 year survival was highly significant (P<.00I): % survival = 1.4637 PHM - 29; r=.99. The survival without death or transplant in patients without HCC was correlated (P < .(J01) with the PMH at 1 year (% survival = 1.18 PHM-29) and 3 years (% survival = 1.25 PHM-391. CONCLUSIONS: 11 The PHM measured by LSS was closely correlated with prognosis and 2) patients wire died with a normal PHM generally died of non-CLD related problems. 2
23.1 9.1 9.1
54.2 59.3 33.3 10.0 33.3 29.2
743 61.8 57.1