Mechanism of the abnormal vitamin K-dependent gamma-carboxylation process in human hepatocellular carcinomas.
BACKGROUND
An important marker for hepatocellular carcinoma is the presence of des-gamma-carboxy (abnormal) prothrombin. However, the molecular basis for the reduced carboxylation of prothrombin is unknown.


METHODS
Two groups of patients were defined according to the absence (Group I, n = 7) or presence (Group II, n = 8) of des-gamma-carboxy prothrombin. The enzymatic activity of gamma-carboxylase and the total microsomal prothrombin concentration were determined in all tumors. The kinetic parameters for the synthetic peptide Phe-Leu-Glu-Glu-Leu (FLEEL) were measured in eight tumors. The gamma-carboxylase mRNA expression was evaluated by Northern blot analysis in 12 of 15 tumors. In addition, the total vitamin K content (K1, K1 epoxide, and menaquinones 4-10) in 10 tumors was investigated by high performance liquid chromatography.


RESULTS
Concentrations of menaquinones 4-10 were normal in the nontumorous part of the liver but significantly decreased (P = 0.02) in all the tumors (Groups I and II). This decrease was more severe in Group II (P = 0.02). The tumors in Group I had normal or increased gamma-carboxylase activity and increased mRNA expression (P < 0.02) as compared with their nontumorous counterparts. The tumors in Group II were heterogeneous. Five tumors displayed low gamma-carboxylase activity, associated with low mRNA expression in two, whereas two others had high gamma-carboxylase activity and mRNA expression. The concentration of FLEEL at half-maximal velocity was normal in all the tumors examined (Groups I and II), and a relation was found between the level of expression of gamma-carboxylase and the maximal velocity for FLEEL carboxylation in the tumors in Group II (r = 0.98; P < 0.01). The microsomal content of normal prothrombin was within normal limits in all tumors (Groups I and II).


CONCLUSIONS
Tumor vitamin K content has a critical role in the synthesis of des-gamma-carboxy prothrombin. Furthermore, the gamma-carboxylase defect, which is observed in some secreting tumors, is the result of the defective gene expression of a normal enzyme and not the consequence of the presence of a competitive inhibitor. It is possible that a 75% reduction in gamma-carboxylase gene expression could take a part in the secretion of des-gamma-carboxy prothrombin, but this mechanism is not predominant.
Huisse
M G
MG
Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
Leclercq
M
M
Belghiti
J
J
Flejou
J F
JF
Suttie
J W
JW
Bezeaud
A
A
Stafford
D W
DW
Guillin
M C
MC
D015415:Biomarkers; D019735:Carbon-Carbon Ligases; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D005165:Factor V / Q000032:analysis; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008025:Ligases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008113:Liver Neoplasms / Q000097:blood / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008862:Microsomes, Liver / Q000201:enzymology; D011498:Protein Precursors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011516:Prothrombin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000032:analysis / Q000235:genetics; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics; D014812:Vitamin K / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*; D010837:Vitamin K 1 / Q000031:analogs & derivatives / Q000032:analysis; D024482:Vitamin K 2 / Q000031:analogs & derivatives; D000509:alpha-Fetoproteins / Q000032:analysis
D015415:Biomarkers; D011498:Protein Precursors; D012334:RNA, Neoplasm; D000509:alpha-Fetoproteins; D024482:Vitamin K 2; D014812:Vitamin K; C016186:vitamin K1 oxide; C030814:menatetrenone; C008208:acarboxyprothrombin; D012313:RNA; D010837:Vitamin K 1; D005165:Factor V; D011516:Prothrombin; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase