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Alpha-Fetoprotein
Alpha-Fetoprotein BT Spear, University of Kentucky College of Medicine, Lexington, KY, USA © 2013 Elsevier Inc. All rights reserved.
Glossary Biomarker A characteristic that is measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or a pharmacologic response to a therapeutic intervention (as defined by National Institutes of Health). Hepatocellular carcinoma (HCC) The most common primary cancer of the liver. The most common cause of HCC worldwide is hepatitis B virus.
History Alpha-fetoprotein (AFP) is a major protein in the serum and amniotic fluid of developing mammalian fetuses, but is nor mally absent in the serum of adult mammals. AFP was first identified in 1957 as a major glycoprotein in human fetal serum. Interest in AFP grew considerably in the early 1960s when increased serum AFP levels were found to be associated with hepatocellular carcinoma. This led to the characterization of AFP as one of the prototype oncofetal proteins in that it is present during fetal life, absent in normal adult tissues, and reactivated in tumors. Subsequently, elevated maternal serum AFP levels were found to be associated with neural tube defects (NTDs) in the developing fetus. These clinically relevant fea tures of AFP expression have led to considerable interest in the regulation and function of AFP over the past 50 years.
Gene Structure and Regulation AFP belongs to the albumin gene family that is comprised of a small number of related genes that include albumin (Alb), AFP, alpha-albumin (also called afamin (Afm)), AFP-related gene (Arg), and the more distantly related vitamin D-binding pro tein (Dbp; also called Gc). These genes arose from a series of duplications of a primordial gene. The Alb family genes are tightly linked in all mammals where this has been studied; this gene family is found on chromosomes 4 and 5 in humans and mice, respectively. The AFP gene is comprised of 15 exons, the first 14 of which code for the AFP protein; these exons span 19.5 kb in humans and 18.2 kb in mice. The major AFP mes senger RNA (mRNA) transcript is 2.2 kb in length, although shorter transcripts have been observed at different times during liver development and in cancer cell lines; these shorter tran scripts may encode proteins with novel functions. The AFP gene is transcribed at high levels in the yolk sac and fetal liver and at much lower levels in the fetal gut and kidney, leading to high AFP protein levels in the serum and amniotic fluid. There is a dramatic decline both in AFP serum levels after birth, due to loss of the yolk sac, and in AFP expression in embryonic tissues (particularly the liver) during the perinatal period; in mice, a nearly 10 000-fold reduction in liver AFP
Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 1
Hereditary persistence of alpha-fetoprotein (HPAFP) An apparently benign autosomal dominant condition in which AFP continues to be expressed in adult life. Neural tube defect A congenital defect due to the improper closure of the neural tube during embryogenesis; clinical manifestations can vary widely from mild to severe. Oncofetal Pertaining to a gene (or protein) that is normally expressed during embryonic development, absent in the adult during normal conditions, and reactivated in cancer.
mRNA levels is seen in the first 3–4 weeks after birth. Under normal physiological conditions, AFP levels remain very low. AFP expression is transiently reactivated in the liver during regen eration following liver damage. In animal studies, regeneration is initiated experimentally most often by partial hepatectomy (removal of two-thirds of the liver) or by treatment with hepato toxins such as carbon tetrachloride. In these situations, reactivation is transient and the AFP gene becomes silent once regeneration ceases. In addition to this transient AFP reactivation, AFP is frequently reactivated in liver cancer (see below). Regulation of AFP expression has been studied primarily using the mouse gene and, to a lesser extent, the rat gene. Mouse AFP transcription is governed by five distinct cis-acting elements including a promoter (within the first ∼200 bp upstream of exon 1), repressor (roughly 1 kb upstream of exon 1), and three independent enhancers, E1, E2, and E3, located −2.5, −5.0, and 6.5 kb, respectively, upstream of exon 1. E1 and E2 are conserved and likely arose from a duplication event, whereas E3 is unique. The availability of genomic sequence information from different mammals has revealed that these three enhancers are not present in all species; E1 is found only in rodents, E3 is found in most species, including humans, whereas E2 is present in all species that have been studied to date. A number of general and liver-specific transcription factors that interact with the AFP control regions have been identified using tissue culture and biochemical strategies. AFP promoter activity is regulated by hepatocyte nuclear factor 1 (HNF1), CAAT/enhancer binding protein (C/EBP), FoxA (formerly called HNF3), Nkx2.8, fetoprotein transcription factor (FTF), and nuclear factor I (NFI). The repressor region interacts with FoxA and p53. The AFP enhancers are regulated by C/EBP, FoxA, HNF6, and several members of the orphan nuclear receptor family. Studies in mice have identified two additional regulators of AFP called zinc fingers and homeoboxes 2 (Zhx2) and zinc finger and broad complex tramtrack bric-a-brac (BTB) domain containing protein 20 (Zbtb20). A natural mutation in the Zhx2 gene in BALB/cJ mice leads to persistent AFP expres sion in the adult liver. Similarly, a liver-specific knockout of Zbtb20 also results in continued hepatic AFP expression after birth. In both cases, AFP expression in the fetal liver is normal.
doi:10.1016/B978-0-12-374984-0.00039-5
89
90
Alpha-Fetoprotein
These data suggest that Zhx2 and Zbtb20 are negative regula tors involved in AFP repression after birth.
Protein Structure and Function The AFP protein is 609 and 605 amino acids with a predicted molecular mass of 67.3 and 68.6 kDa in humans and mice, respectively. AFP migrates with an apparent molecular mass of 69–73 kDa, depending on the extent of glycosylation. The pro tein contains 15 disulfide bonds and is comprised of three domains that form a V-like structure. The protein is highly conserved across mammals. In addition to the monomeric form, AFP can also multimerize. AFP functions as a serum transport protein. AFP binds numerous molecules, including estrogen, fatty acids, bilirubin, steroids, heavy metals (including copper and nickel), various environmental compounds, and certain drugs; in these capa cities, AFP could potentially control cell proliferation and differentiation in the developing fetus as well as tumor cell growth. In particular, it has been proposed that AFP could regulate the bioavailability of estradiol to the developing fetal brain, and thus influence neurogenesis and/or behavior. The high AFP levels during embryogenesis could also contribute to the osmotic pressure of intravascular fluids. Several studies have suggested that AFP may be immunosuppressive and there fore protect the developing fetus from the maternal immune system. However, several examples of congenital AFP defi ciency have been observed in humans, with no apparent consequences. In addition, mice have been generated in which the AFP gene has been deleted by homologous recom bination in embryonic stem (ES) cells; mice that are deficient in AFP develop normally and are viable. Thus, despite its high levels, AFP is not essential for fetal development. This does not necessarily mean that AFP does not carry out the functions described above; it may be that these functions are redundant and can be performed by other proteins, that is, other members of the Alb family that are also expressed during fetal life. More recently, a careful analysis has revealed physiological and behavioral changes in female mice that lack AFP. First, AFP-deficient female mice are sterile due to a lack of ovulation. This defect is not intrinsic to the ovaries, but is rather due to an inadequate hormonal environment that is necessary for ovulation to occur normally. This finding implicates the hypothalamus–pituitary–gonadal axis in this defect. Indeed, numerous genes expressed in the pituitary and hypothalamus are disregulated in AFP-deficient female mice. Furthermore, the defeminization of the brain in AFP-deficient female mice also results in anomalies in sexual behavior, including reduced sexual receptivity and reduced maternal care. Based on these studies, it appears that AFP protects the developing female mouse brain from the effects of estradiol (which promotes masculinization). It is not known if AFP has a similar effect in other species, although it should be noted that human AFP does not bind estrogen.
cancer worldwide (although the incidence of HCC varies greatly in different populations). Hepatitis B virus (HBV) infection is the most common cause of HCC, although other risk factors include hepatitis C virus (HCV), infection alcohol-induced cirrhosis, metabolic disorders (i.e., Wilson’s disease), and aflatoxins (in certain geographical regions). Increased serum AFP has also been associated with germ cell tumors (embryonal carcinoma and teratomas) and, to a lesser extent, with pancreatic, stomach, and kidney tumors. Activated AFP levels can also be associated with non-neoplastic liver disease, including viral hepatitis as well as drug- or alcohol-induced liver damage. Fetal-derived AFP can cross the placental barrier and be found in the maternal serum at measureable levels. In the mid-1970s, elevated maternal AFP levels were found to be associated with fetuses having NTDs. This led to maternal AFP being used as a diagnostic marker for NTDs such as spina bifida and anencephaly, as well as other fetal abnormalities. By contrast, abnormally low maternal AFP levels have been asso ciated with chromosomal abnormalities, including trisomy 21 (Down’s syndrome). Maternal AFP screening is recommended more frequently in older pregnant women, since the likelihood of these conditions increases with maternal age.
Future Considerations AFP is among the most widely used diagnostic biomarkers, based on its use for screening for malignancies and prenatal abnormalities. However, there are limitations in the use of AFP as a tumor and prenatal biomarker. For example, AFP levels are not always elevated in HCC, and AFP can be elevated during liver disease in which cancer is not present. This has led to a search for other biomarkers and the use of imaging technologies to augment and/or replace AFP screening. For example, recent studies suggest that Glypican 3 may be a better marker than AFP for HCC screening. Also, it should be noted that the widespread screening for AFP has identified several families in which there is an incomplete shut off of AFP at birth, a benign condition termed hereditary persistence of AFP (HPAFP). Therefore, AFP screening as a diagnostic tool for cancers or birth defects must be interpreted with caution. Nonetheless, AFP is likely to remain an important biomarker for physicians, and further studies are needed to determine whether AFP has a functional role in these processes. There is also interest in using AFP (and AFP peptides) in anticancer treatments. AFP is expressed in liver stem cells, and there is considerable interest in these types of cells for the treatment of liver disease. Furthermore, a better understanding of AFP regulation will elucidate mechanisms of gene regulation during development and disease.
See also: Down Syndrome; Germ Cell; Gene Regulation.
Further Reading Clinical Significance AFP is of considerable clinical interest as a diagnostic marker. Serum AFP levels are often elevated in the serum of patients with hepatocellular carcinoma (HCC), which is the fifth most common
Abelev GI and Eraiser TL (1999) Cellular aspects of alpha-fetoprotein re-expression in tumors. Seminars in Cancer Biology 9: 95–107. Cunniff C and American Academy of Pediatrics Committee on Genetics (2004) Prenatal screening and diagnosis for pediatricians. Pediatrics 114: 889–894. De Mees C, Bakker J, Szpirer J, and Szpirer C (2007) Alpha-fetoprotein: From a diagnostic biomarker to a key role in female fertility. Biomarker Insights 1: 82–85.
Alpha-Fetoprotein Leighton PC, Kitau MJ, Chard T, Gordon YB, and Leek AE (1975) Levels of alpha-fetoprotein in maternal blood as a screening test for fetal neural-tube defects. Lancet 2: 1012–1015. Mizejewski GJ (2001) Alpha-fetoprotein structure and function: Relevance to isoforms, epitopes, and conformational variants. Experimental Biology and Medicine (Maywood) 226: 377–408. Mizejewski GJ (2004) Biological roles of alpha-fetoprotein during pregnancy and perinatal development. Experimental Biology and Medicine (Maywood) 229: 439–463. Sell S (2008) Alpha-fetoprotein, stem cells and cancer: How study of the production of alpha-fetoprotein during chemical hepatocarcinogenesis led to reaffirmation of the stem cell theory of cancer. Tumor Biology 29: 161–180.
91
Spear BT (1999) Alpha-fetoprotein gene regulation: Lessons from transgenic mice. Seminars in Cancer Biology 9: 109–116.
Relevant Websites http://genome.ucsc.edu – AFP gene/genomic organization on the UCSC genome. http://www.ncbi.nlm.nih.gov – AFP on the OMIM (On-line Mendelian Inheritance in Man) site. http://atlasgeneticsoncology.org – AFP structure/function studies.
Glossary Biomarker A characteristic that is measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or a pharmacologic response to a therapeutic intervention (as defined by National Institutes of Health). Hepatocellular carcinoma (HCC) The most common primary cancer of the liver. The most common cause of HCC worldwide is hepatitis B virus.
History Alpha-fetoprotein (AFP) is a major protein in the serum and amniotic fluid of developing mammalian fetuses, but is nor mally absent in the serum of adult mammals. AFP was first identified in 1957 as a major glycoprotein in human fetal serum. Interest in AFP grew considerably in the early 1960s when increased serum AFP levels were found to be associated with hepatocellular carcinoma. This led to the characterization of AFP as one of the prototype oncofetal proteins in that it is present during fetal life, absent in normal adult tissues, and reactivated in tumors. Subsequently, elevated maternal serum AFP levels were found to be associated with neural tube defects (NTDs) in the developing fetus. These clinically relevant fea tures of AFP expression have led to considerable interest in the regulation and function of AFP over the past 50 years.
Gene Structure and Regulation AFP belongs to the albumin gene family that is comprised of a small number of related genes that include albumin (Alb), AFP, alpha-albumin (also called afamin (Afm)), AFP-related gene (Arg), and the more distantly related vitamin D-binding pro tein (Dbp; also called Gc). These genes arose from a series of duplications of a primordial gene. The Alb family genes are tightly linked in all mammals where this has been studied; this gene family is found on chromosomes 4 and 5 in humans and mice, respectively. The AFP gene is comprised of 15 exons, the first 14 of which code for the AFP protein; these exons span 19.5 kb in humans and 18.2 kb in mice. The major AFP mes senger RNA (mRNA) transcript is 2.2 kb in length, although shorter transcripts have been observed at different times during liver development and in cancer cell lines; these shorter tran scripts may encode proteins with novel functions. The AFP gene is transcribed at high levels in the yolk sac and fetal liver and at much lower levels in the fetal gut and kidney, leading to high AFP protein levels in the serum and amniotic fluid. There is a dramatic decline both in AFP serum levels after birth, due to loss of the yolk sac, and in AFP expression in embryonic tissues (particularly the liver) during the perinatal period; in mice, a nearly 10 000-fold reduction in liver AFP
Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 1
Hereditary persistence of alpha-fetoprotein (HPAFP) An apparently benign autosomal dominant condition in which AFP continues to be expressed in adult life. Neural tube defect A congenital defect due to the improper closure of the neural tube during embryogenesis; clinical manifestations can vary widely from mild to severe. Oncofetal Pertaining to a gene (or protein) that is normally expressed during embryonic development, absent in the adult during normal conditions, and reactivated in cancer.
mRNA levels is seen in the first 3–4 weeks after birth. Under normal physiological conditions, AFP levels remain very low. AFP expression is transiently reactivated in the liver during regen eration following liver damage. In animal studies, regeneration is initiated experimentally most often by partial hepatectomy (removal of two-thirds of the liver) or by treatment with hepato toxins such as carbon tetrachloride. In these situations, reactivation is transient and the AFP gene becomes silent once regeneration ceases. In addition to this transient AFP reactivation, AFP is frequently reactivated in liver cancer (see below). Regulation of AFP expression has been studied primarily using the mouse gene and, to a lesser extent, the rat gene. Mouse AFP transcription is governed by five distinct cis-acting elements including a promoter (within the first ∼200 bp upstream of exon 1), repressor (roughly 1 kb upstream of exon 1), and three independent enhancers, E1, E2, and E3, located −2.5, −5.0, and 6.5 kb, respectively, upstream of exon 1. E1 and E2 are conserved and likely arose from a duplication event, whereas E3 is unique. The availability of genomic sequence information from different mammals has revealed that these three enhancers are not present in all species; E1 is found only in rodents, E3 is found in most species, including humans, whereas E2 is present in all species that have been studied to date. A number of general and liver-specific transcription factors that interact with the AFP control regions have been identified using tissue culture and biochemical strategies. AFP promoter activity is regulated by hepatocyte nuclear factor 1 (HNF1), CAAT/enhancer binding protein (C/EBP), FoxA (formerly called HNF3), Nkx2.8, fetoprotein transcription factor (FTF), and nuclear factor I (NFI). The repressor region interacts with FoxA and p53. The AFP enhancers are regulated by C/EBP, FoxA, HNF6, and several members of the orphan nuclear receptor family. Studies in mice have identified two additional regulators of AFP called zinc fingers and homeoboxes 2 (Zhx2) and zinc finger and broad complex tramtrack bric-a-brac (BTB) domain containing protein 20 (Zbtb20). A natural mutation in the Zhx2 gene in BALB/cJ mice leads to persistent AFP expres sion in the adult liver. Similarly, a liver-specific knockout of Zbtb20 also results in continued hepatic AFP expression after birth. In both cases, AFP expression in the fetal liver is normal.
doi:10.1016/B978-0-12-374984-0.00039-5
89
90
Alpha-Fetoprotein
These data suggest that Zhx2 and Zbtb20 are negative regula tors involved in AFP repression after birth.
Protein Structure and Function The AFP protein is 609 and 605 amino acids with a predicted molecular mass of 67.3 and 68.6 kDa in humans and mice, respectively. AFP migrates with an apparent molecular mass of 69–73 kDa, depending on the extent of glycosylation. The pro tein contains 15 disulfide bonds and is comprised of three domains that form a V-like structure. The protein is highly conserved across mammals. In addition to the monomeric form, AFP can also multimerize. AFP functions as a serum transport protein. AFP binds numerous molecules, including estrogen, fatty acids, bilirubin, steroids, heavy metals (including copper and nickel), various environmental compounds, and certain drugs; in these capa cities, AFP could potentially control cell proliferation and differentiation in the developing fetus as well as tumor cell growth. In particular, it has been proposed that AFP could regulate the bioavailability of estradiol to the developing fetal brain, and thus influence neurogenesis and/or behavior. The high AFP levels during embryogenesis could also contribute to the osmotic pressure of intravascular fluids. Several studies have suggested that AFP may be immunosuppressive and there fore protect the developing fetus from the maternal immune system. However, several examples of congenital AFP defi ciency have been observed in humans, with no apparent consequences. In addition, mice have been generated in which the AFP gene has been deleted by homologous recom bination in embryonic stem (ES) cells; mice that are deficient in AFP develop normally and are viable. Thus, despite its high levels, AFP is not essential for fetal development. This does not necessarily mean that AFP does not carry out the functions described above; it may be that these functions are redundant and can be performed by other proteins, that is, other members of the Alb family that are also expressed during fetal life. More recently, a careful analysis has revealed physiological and behavioral changes in female mice that lack AFP. First, AFP-deficient female mice are sterile due to a lack of ovulation. This defect is not intrinsic to the ovaries, but is rather due to an inadequate hormonal environment that is necessary for ovulation to occur normally. This finding implicates the hypothalamus–pituitary–gonadal axis in this defect. Indeed, numerous genes expressed in the pituitary and hypothalamus are disregulated in AFP-deficient female mice. Furthermore, the defeminization of the brain in AFP-deficient female mice also results in anomalies in sexual behavior, including reduced sexual receptivity and reduced maternal care. Based on these studies, it appears that AFP protects the developing female mouse brain from the effects of estradiol (which promotes masculinization). It is not known if AFP has a similar effect in other species, although it should be noted that human AFP does not bind estrogen.
cancer worldwide (although the incidence of HCC varies greatly in different populations). Hepatitis B virus (HBV) infection is the most common cause of HCC, although other risk factors include hepatitis C virus (HCV), infection alcohol-induced cirrhosis, metabolic disorders (i.e., Wilson’s disease), and aflatoxins (in certain geographical regions). Increased serum AFP has also been associated with germ cell tumors (embryonal carcinoma and teratomas) and, to a lesser extent, with pancreatic, stomach, and kidney tumors. Activated AFP levels can also be associated with non-neoplastic liver disease, including viral hepatitis as well as drug- or alcohol-induced liver damage. Fetal-derived AFP can cross the placental barrier and be found in the maternal serum at measureable levels. In the mid-1970s, elevated maternal AFP levels were found to be associated with fetuses having NTDs. This led to maternal AFP being used as a diagnostic marker for NTDs such as spina bifida and anencephaly, as well as other fetal abnormalities. By contrast, abnormally low maternal AFP levels have been asso ciated with chromosomal abnormalities, including trisomy 21 (Down’s syndrome). Maternal AFP screening is recommended more frequently in older pregnant women, since the likelihood of these conditions increases with maternal age.
Future Considerations AFP is among the most widely used diagnostic biomarkers, based on its use for screening for malignancies and prenatal abnormalities. However, there are limitations in the use of AFP as a tumor and prenatal biomarker. For example, AFP levels are not always elevated in HCC, and AFP can be elevated during liver disease in which cancer is not present. This has led to a search for other biomarkers and the use of imaging technologies to augment and/or replace AFP screening. For example, recent studies suggest that Glypican 3 may be a better marker than AFP for HCC screening. Also, it should be noted that the widespread screening for AFP has identified several families in which there is an incomplete shut off of AFP at birth, a benign condition termed hereditary persistence of AFP (HPAFP). Therefore, AFP screening as a diagnostic tool for cancers or birth defects must be interpreted with caution. Nonetheless, AFP is likely to remain an important biomarker for physicians, and further studies are needed to determine whether AFP has a functional role in these processes. There is also interest in using AFP (and AFP peptides) in anticancer treatments. AFP is expressed in liver stem cells, and there is considerable interest in these types of cells for the treatment of liver disease. Furthermore, a better understanding of AFP regulation will elucidate mechanisms of gene regulation during development and disease.
See also: Down Syndrome; Germ Cell; Gene Regulation.
Further Reading Clinical Significance AFP is of considerable clinical interest as a diagnostic marker. Serum AFP levels are often elevated in the serum of patients with hepatocellular carcinoma (HCC), which is the fifth most common
Abelev GI and Eraiser TL (1999) Cellular aspects of alpha-fetoprotein re-expression in tumors. Seminars in Cancer Biology 9: 95–107. Cunniff C and American Academy of Pediatrics Committee on Genetics (2004) Prenatal screening and diagnosis for pediatricians. Pediatrics 114: 889–894. De Mees C, Bakker J, Szpirer J, and Szpirer C (2007) Alpha-fetoprotein: From a diagnostic biomarker to a key role in female fertility. Biomarker Insights 1: 82–85.
Alpha-Fetoprotein Leighton PC, Kitau MJ, Chard T, Gordon YB, and Leek AE (1975) Levels of alpha-fetoprotein in maternal blood as a screening test for fetal neural-tube defects. Lancet 2: 1012–1015. Mizejewski GJ (2001) Alpha-fetoprotein structure and function: Relevance to isoforms, epitopes, and conformational variants. Experimental Biology and Medicine (Maywood) 226: 377–408. Mizejewski GJ (2004) Biological roles of alpha-fetoprotein during pregnancy and perinatal development. Experimental Biology and Medicine (Maywood) 229: 439–463. Sell S (2008) Alpha-fetoprotein, stem cells and cancer: How study of the production of alpha-fetoprotein during chemical hepatocarcinogenesis led to reaffirmation of the stem cell theory of cancer. Tumor Biology 29: 161–180.
91
Spear BT (1999) Alpha-fetoprotein gene regulation: Lessons from transgenic mice. Seminars in Cancer Biology 9: 109–116.
Relevant Websites http://genome.ucsc.edu – AFP gene/genomic organization on the UCSC genome. http://www.ncbi.nlm.nih.gov – AFP on the OMIM (On-line Mendelian Inheritance in Man) site. http://atlasgeneticsoncology.org – AFP structure/function studies.