In this paper, we explore the role played by apoptosis in the genesis of cancer. The hypothesis under test is that apoptosis affords a means whereby cells that have sustained genotoxic damage can be irreversibly deleted from the generative compartment of the affected tissue. Such damaged cells might otherwise have acquired serious but survivable DNA modifications, and so could have become the precursors of malignant clones (Fig. 1). One means of testing the hypothesis is to devise systems in which a critical gene on the signaling pathway from genotoxic damage to apoptosis is selectively disabled. Following exposure to the genotoxic agent, cells bearing such disabled genes would be expected to survive, generating a population of previously "forbidden" cells. The hypothesis predicts that these cells should have undergone genomic changes similar or identical to those known to be clonally expanded in cancer, a proposition that can be addressed directly.
Early axon tracts in the developing vertebrate brain are established along precise paths. Yet, little is known about axon guidance processes at early stages of rostral brain development. Using whole mount in situ hybridisation in combination with immunohistochemistry, we have analysed the expression patterns of Slits, Netrins, Semaphorins and the respective receptors during the formation of the early axon scaffold, particularly focusing on the pretectal-mesencephalic boundary. Many of these guidance molecules are expressed in close correlation with the growing tracts, and the nuclei of the corresponding neurons often express the respective receptors. The expression patterns of Slits and Netrins implicate them with the positioning of the longitudinal tracts along the dorsoventral axis, while Semaphorins could provide guidance at specific choice points. Our study provides a catalogue of gene expression for future studies on axon guidance mechanisms in the early brain.
Neutral lipid is stored in spherical organelles called lipid droplets that are bounded by a coat of proteins. The protein that is most frequently found at the surface of lipid droplets is adipocyte differentiation-related protein (ADRP). In this study, we demonstrate that fusion of either the human or mouse ADRP coding sequences to green fluorescent protein (GFP) does not disrupt the ability of the protein to associate with lipid droplets. Using this system to identify targeting elements, discontinuous segments within the coding region were required for directing ADRP to lipid droplets. GFP-tagged protein was employed also to examine the behavior of lipid droplets in live cells. Time lapse microscopy demonstrated that in HuH-7 cells, which are derived from a human hepatoma, a small number of lipid droplets could move rapidly, indicating transient association with intracellular transport pathways. Most lipid droplets did not show such movement but oscillated within a confined area; these droplets were in close association with the endoplasmic reticulum membrane and moved in concert with the endoplasmic reticulum. Fluorescence recovery analysis of GFP-tagged ADRP in live cells revealed that surface proteins do not rapidly diffuse between lipid droplets, even in conditions where they are closely packed. This system provides new insights into the properties of lipid droplets and their interaction with cellular processes. Neutral lipid is stored in spherical organelles called lipid droplets that are bounded by a coat of proteins. The protein that is most frequently found at the surface of lipid droplets is adipocyte differentiation-related protein (ADRP). In this study, we demonstrate that fusion of either the human or mouse ADRP coding sequences to green fluorescent protein (GFP) does not disrupt the ability of the protein to associate with lipid droplets. Using this system to identify targeting elements, discontinuous segments within the coding region were required for directing ADRP to lipid droplets. GFP-tagged protein was employed also to examine the behavior of lipid droplets in live cells. Time lapse microscopy demonstrated that in HuH-7 cells, which are derived from a human hepatoma, a small number of lipid droplets could move rapidly, indicating transient association with intracellular transport pathways. Most lipid droplets did not show such movement but oscillated within a confined area; these droplets were in close association with the endoplasmic reticulum membrane and moved in concert with the endoplasmic reticulum. Fluorescence recovery analysis of GFP-tagged ADRP in live cells revealed that surface proteins do not rapidly diffuse between lipid droplets, even in conditions where they are closely packed. This system provides new insights into the properties of lipid droplets and their interaction with cellular processes. adipocyte differentiation-related protein human ADRP mouse ADRP enhanced green fluorescent protein enhanced yellow fluorescent protein open reading frame phosphate-buffered saline reverse transcriptase hepatitis C virus fluorescent recovery after photobleaching In mammalian cells, lipid droplets serve as storage organelles, consisting primarily of cholesterol ester and triacylglyerols (reviewed in Ref. 1Murphy D.J. Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google Scholar). Although long considered to be inert structures, there is now increasing evidence that they play active and diverse roles in the life cycle of cells. Recently, they have been implicated in maintenance of intracellular cholesterol balance and transport of lipids through association with caveolin proteins (2Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Crossref PubMed Scopus (274) Google Scholar). Moreover, the lipid stored in droplets is utilized for specialized purposes in certain cell types. For example, steroid hormones in steroidogenic cells are produced from cholesterol ester stored in lipid droplets (3Freeman D.A. Ascoli M. J. Biol. Chem. 1982; 257: 14231-14238Abstract Full Text PDF PubMed Google Scholar); mammary epithelial cells release milk fat globules from their apical surface that are directly derived from aggregated lipid droplets (4Mather I.H. Keenan T.W. J. Mammary Gland Biol. Neoplasia. 1998; 3: 259-273Crossref PubMed Scopus (282) Google Scholar); lipid droplets appear to be the primary source of fatty acids that are converted into triacylglycerols and incorporated into very low density lipoprotein in hepatocytes (5Gibbons G.F. Islam K. Pease R.J. Biochim. Biophys. Acta. 2000; 1483: 37-57Crossref PubMed Scopus (248) Google Scholar). There is also a correlation between particular human diseases and accumulation of lipid droplets that suggests they are markers of pathological changes. Such diseases include atheroma, steatosis, obesity, and some cancers (6Heid H.W. Moll R. Schwetlick I. Rackwitz H.R. Keenan T.W. Cell Tissue Res. 1998; 294: 309-321Crossref PubMed Scopus (356) Google Scholar, 7Murphy D.J. Vance J. Trends Biol. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). More recently, it has been suggested that aberrant targeting of the Nir2 protein to lipid droplets may induce changes to lipid transport that could correlate with retinal degeneration seen inDrosophila mutants (8Litvak V. Shaul Y.D. Shulewitz M. Amarilio R. Carmon S. Lev S. Curr. Biol. 2002; 12: 1513-1518Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Indeed, lipid droplets may have important functions in viral and parasitic infections (9Barba G. Harper F. Harada T. Kohara M. Goulinet S. Matsuura Y. Eder G. Schaff Zs. Chapman M.J. Miyamura T. Brechot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1200-1205Crossref PubMed Scopus (573) Google Scholar, 10Charron A.J. Sibley L.D. J. Cell Sci. 2002; 115: 3049-3059Crossref PubMed Google Scholar, 11Hope R.G. McLauchlan J. J. Gen. Virol. 2000; 81: 1913-1925Crossref PubMed Scopus (192) Google Scholar, 12Hope R.G. Murphy D.J. McLauchlan J. J. Biol. Chem. 2002; 277: 4261-4270Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13Moriya K. Yotsuyanagi H. Shintani Y. Fujie H. Ishibashi K. Matsuura Y. Miyamura T. Koike K. J. Gen. Virol. 1997; 78: 1527-1531Crossref PubMed Scopus (576) Google Scholar).The surface of lipid droplets has a proteinaceous layer that is thought to prevent fusion with any adjacent lipophilic surface. However, analysis of the properties of surface proteins has received only limited attention. The most widely characterized lipid droplet-associated proteins are the perilipins, a family of polypeptides generated by alternative splicing from a single copy gene (14Lu X.Y. Gruia-Gray J. Copeland N.G. Gilbert D.J. Jenkins N.A. Londos C. Kimmel A.R. Mamm. Genome. 2001; 12: 741-749Crossref PubMed Scopus (181) Google Scholar), and adipocyte differentiation-related protein (ADRP,1 also termed adipophilin) (6Heid H.W. Moll R. Schwetlick I. Rackwitz H.R. Keenan T.W. Cell Tissue Res. 1998; 294: 309-321Crossref PubMed Scopus (356) Google Scholar, 15Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar, 16Gao J. Ye H. Serrero G. J. Cell. Physiol. 2000; 182: 297-302Crossref PubMed Scopus (96) Google Scholar, 17Jiang H.P. Serrero G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7856-7860Crossref PubMed Scopus (226) Google Scholar). Expression of the perilipins appears restricted to adipocytes and steroidogenic cells (Refs. 18Greenberg A.S. Egan J.J. Wek S.A. Moos M.C. Londos C. Kimmel A.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 12035-12039Crossref PubMed Scopus (211) Google Scholar and 19Servetnick D.A. Brasaemle D.L. Gruia-Gray J. Kimmel A.R. Wolff J. Londos C. J. Biol. Chem. 1995; 269: 16970-16973Abstract Full Text Full Text PDF Scopus (162) Google Scholar, reviewed in Ref. 20Londos C. Brasaemle D.L. Schultz C.J. Segrest J.P. Kimmel A.R. Semin. Cell. Dev. Biol. 1999; 10: 51-58Crossref PubMed Scopus (365) Google Scholar) whereas ADRP is detected in a broad range of different tissues (15Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar). Examination of a variety of tissue culture cells also has revealed ADRP as a ubiquitous component of lipid droplets (6Heid H.W. Moll R. Schwetlick I. Rackwitz H.R. Keenan T.W. Cell Tissue Res. 1998; 294: 309-321Crossref PubMed Scopus (356) Google Scholar, 15Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar). Moreover, enhanced expression of ADRP is a useful marker for pathologies that are characterized by increased accumulations of lipid droplets (6Heid H.W. Moll R. Schwetlick I. Rackwitz H.R. Keenan T.W. Cell Tissue Res. 1998; 294: 309-321Crossref PubMed Scopus (356) Google Scholar, 21Buechler C. Ritter M. Duong C.Q. Orso E. Kapinsky M. Schmitz G. Biochim. Biophys. Acta. 2001; 1532: 97-104Crossref PubMed Scopus (81) Google Scholar, 22Rae F.K. Stephenson S.A. Nicol D.L. Clements J.A. Int. J. Cancer. 2000; 88: 726-732Crossref PubMed Scopus (114) Google Scholar, 23Steiner S. Wahl D. Mangold B.L.K. Robison R. Raymackers J. Meheus L. Anderson N.L. Cordier A. Biochem. Biophys. Res. Commun. 1996; 218: 777-782Crossref PubMed Scopus (62) Google Scholar, 24Wang X.K. Reape T.J. Li X. Rayner K. Webb C.L. Burnand K.G. Lysko P.G. FEBS Lett. 1999; 462: 145-150Crossref PubMed Scopus (88) Google Scholar). Hence, analysis of ADRP would provide valuable insight into the nature of proteins that associate with lipid droplets. In addition, ADRP offers potential for describing the characteristics of lipid droplets.In this study, we have fused green and yellow fluorescent proteins (EGFP and EYFP) to the human and mouse ADRP coding sequences and examined the ability of the fusion proteins to associate with lipid droplets in tissue culture cells. This system was employed to identify sequences in ADRP that are required for localization to lipid droplets. ADRP tagged with fluorescent proteins also was used to analyze the behavior of lipid droplets in live cells and their interaction with other intracellular compartments.DISCUSSIONADRP is a component of lipid droplets found in a broad range of cultured cells and tissues. Accordingly, it is a useful model protein to examine the properties of proteinaceous components of lipid droplets and the innate behavior of these organelles. In our study, we have employed fusion of both human and mouse forms of ADRP with EGFP to study both of these aspects.Analysis of the sequences required to direct hADRP to lipid droplets revealed that targeting signals are distributed throughout the polypeptide coding region (Fig. 8). In the context of our study, we define targeting as the process through which ADRP is directed to lipid droplets and then forms a stable association with the organelles. Therefore, reduced targeting efficiency could result from the inability of the tagged proteins to attach stably to lipid droplets, possibly through competition with endogenous ADRP. We found that removal of the N-terminal 28 amino acids impaired lipid droplet localization and a second targeting element was located between residues 139 and 220. Sequences that can contribute to efficient association with lipid droplets also were present in the C-terminal region from amino acids 221 to 437 but this segment of the protein alone was not capable of directing the protein to droplets. These regions of the protein do not form a contiguous targeting element because sequences between amino acids 61 and 130 can be removed with no significant impact on the efficiency of lipid droplet association. Our results indicate that removing one of these targeting sequences can impair but not abolish lipid droplet localization, suggesting that there is redundancy in the sequence requirements for efficient association. Thus, identification of indispensable sequences has not been possible. A recent study also indicated redundancy within the sequences that were necessary for directing perilipin to lipid droplets (28Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Moreover, simple targeting signals for peroxisomal membrane proteins have not been readily identified and it appears that more than one targeting element may reside within such proteins (29Jones J.M. Morrell J.C. Gould S.J. J. Cell Biol. 2001; 153: 1141-1150Crossref PubMed Scopus (90) Google Scholar). Therefore, sequence redundancy may be a general feature for directing proteins to the surface of organelles such as peroxisomes and lipid droplets.It has been proposed that ADRP can be separated into two domains, PAT-1 and PAT-2, based on sequence identity with other lipid droplet-associated proteins (14Lu X.Y. Gruia-Gray J. Copeland N.G. Gilbert D.J. Jenkins N.A. Londos C. Kimmel A.R. Mamm. Genome. 2001; 12: 741-749Crossref PubMed Scopus (181) Google Scholar). PAT-1 is considered to consist of approximately the N-terminal 100 amino acids of the protein and the remaining C-terminal segment is PAT-2 (Fig. 8). Sequence identity between PAT-1 domains for lipid droplet proteins is greater than that for PAT-2. Indeed, using PAT-1 sequences, lipid droplet proteins have been identified putatively in Drosophila melanogaster andBombyx mori (14Lu X.Y. Gruia-Gray J. Copeland N.G. Gilbert D.J. Jenkins N.A. Londos C. Kimmel A.R. Mamm. Genome. 2001; 12: 741-749Crossref PubMed Scopus (181) Google Scholar). Our analysis shows that the N-terminal 28 amino acids of hADRP, which are necessary for efficient targeting, are contained within PAT-1. However, the other sequences that have been identified as important for efficient lipid droplet association are located in PAT-2. The region within perilipin that has been identified as important for localization to lipid droplets also lies in PAT-2 (28Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). This indicates that the higher sequence identity in PAT-1 between lipid droplet-associated proteins such as ADRP and perilipin does not necessarily represent conserved targeting sequences. Hence, the sequences that direct these proteins to lipid droplets may be distinct. It has also been observed that a region in the PAT-1 domain of ADRP has sequence identity with a tandem repeat in the plasma membrane protein, S3–12 (20Londos C. Brasaemle D.L. Schultz C.J. Segrest J.P. Kimmel A.R. Semin. Cell. Dev. Biol. 1999; 10: 51-58Crossref PubMed Scopus (365) Google Scholar, 30Scherer P.E. Bickel P.E. Kotler M. Lodish H.F. Nat. Biotechnol. 1998; 16: 581-586Crossref PubMed Scopus (107) Google Scholar). Our results show that removing this segment from ADRP has no effect on lipid droplet association (Fig. 8), leading us to conclude that any sequence identity with this tandem repeat is not related to targeting of ADRP.The discontinuous nature of the targeting sequences in ADRP differs from the characteristics of other lipid droplet-associated proteins apart from perilipin. Association of HCV core protein with lipid droplets requires a domain of about 55 amino acids that is present also in a related virus, GB virus-B but not in pesti- and flaviviruses that share the same genomic organization as HCV (11Hope R.G. McLauchlan J. J. Gen. Virol. 2000; 81: 1913-1925Crossref PubMed Scopus (192) Google Scholar, 12Hope R.G. Murphy D.J. McLauchlan J. J. Biol. Chem. 2002; 277: 4261-4270Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Apart from a short stretch of amino acids, removal or substitution of amino acids along the length of the domain abolishes lipid droplet localization. In addition, plant oleosins have a central region of 85 amino acids that has all of the sequences required for efficient attachment in mammalian and plant cells; this central region is flanked by domains that appear to be dispensable for lipid droplet association (12Hope R.G. Murphy D.J. McLauchlan J. J. Biol. Chem. 2002; 277: 4261-4270Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 31van Rooijen G.J.H. Moloney M.M. Plant Physiol. 1995; 109: 1353-1361Crossref PubMed Scopus (58) Google Scholar). Moreover, the targeting sequences in the HCV core and plant oleosin proteins are hydrophobic, whereas no corresponding segments with similar characteristics are found in either human or mouse ADRP. This suggests that processes, which are different from those for the viral and plant proteins, may guide localization of ADRP to lipid droplets. In the case of HCV core, trafficking of the protein to lipid droplets involves initial targeting and cleavage of a precursor protein at the ER and core is found both at the ER membrane as well as at the surface of lipid droplets (32McLauchlan J. Lemberg M.K. Hope R.G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (387) Google Scholar). It is likely that transfer of core between the two organelles is facilitated by the attachment of lipid droplets to the ER, which we describe from our live cell studies. However, for ADRP, no staining of the ER is found by either indirect immunofluorescence or live cell analysis. The mechanism that controls partitioning of ADRP is presumably a reflection of differences between either components or compositions of lipid droplets and the ER that favor interaction with lipid droplets. In addition, the literature indicates that ADRP is synthesized on free and not ER-bound polyribosomes (20Londos C. Brasaemle D.L. Schultz C.J. Segrest J.P. Kimmel A.R. Semin. Cell. Dev. Biol. 1999; 10: 51-58Crossref PubMed Scopus (365) Google Scholar). A similar situation has been demonstrated for the perilipins (33Brasaemle D.L. Barber T. Kimmel A.R. Londos C. J. Biol. Chem. 1997; 272: 9378-9387Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). It may be that association of ADRP and perilipin with the ER is not favored to prevent fusion of lipid droplets with the ER after their formation.Lipid droplets often occur as clusters in a number of cell types. This may indicate that they form a connected network in which transfer of macromolecules between droplets could occur. FRAP is gaining wide use to determine the mobility of GFP-tagged proteins in organelles with continuous surfaces such as the ER, Golgi, and plasma membranes (Refs.34Cole N.B. Smith C.L. Sciaky N. Terasaki M. Edidin M. Lippincott-Schwartz J. Science. 1996; 273: 797-801Crossref PubMed Scopus (403) Google Scholar, 35Nehls S. Snapp E.L. Cole N.B. Zaal K.J.M. Kenworthy A.K. Roberts T.H. Ellenberg J. Presley J.F. Siggia E. Lippincott-Schwartz J. Nat. Cell Biol. 2000; 2: 288-295Crossref PubMed Scopus (215) Google Scholar, 36Niv H. Gutman O. Kloog Y. Henis Y.I. J. Cell Biol. 2002; 157: 865-872Crossref PubMed Scopus (192) Google Scholar, reviewed in Ref. 37Reits E.A.J. Neefjes J.J. Nat. Cell Biol. 2001; 3: E145-E147Crossref PubMed Scopus (503) Google Scholar). More recently, FRAP has revealed that clusters of mitochondria are functionally distinct and unconnected (38Collins T.J. Berridge M.J. Lipp P. Bootman M.D. EMBO J. 2002; 21: 1616-1627Crossref PubMed Scopus (459) Google Scholar). From our data, ADRP does not rapidly diffuse between lipid droplets that are apparently in contact with each other. This suggests that droplets exist as discrete entities and that there is little or no transfer of surface proteins between them. As a consequence, there may be little turnover of ADRP at the surface of lipid droplets after their formation.Analysis of live cells offers advantages for determining the characteristics of lipid droplets. Lipid droplets are heterogeneous in size both between different cell types and even in individual cells. The factors that govern their size are not known but could be determined during their biogenesis. It is also possible that additional neutral lipid is added after formation, either through transfer from the ER or from other lipid droplets. In HuH-7 cells, lipid droplets were no greater than about 1 μm in diameter in live cells using GFP-ADRP as a marker. However, upon fixation and subsequent treatment with alcohol to stain lipid droplets with oil red O, their maximum size increased to 3 μm (data not shown). We propose that this discrepancy results from fusion of closely associated droplets and the size distribution in cells, which are stained using alcoholic solutions, is not authentic. Such an observation may have a bearing on the interpretation of lipid droplet size in cells that store large quantities of neutral lipid and in pathological conditions where lipid storage is assessed. For example, steatosis of the liver can be described as either micro- or macrovesicular depending on the diameter of lipid droplets (39Burt A.D. Mutton A. Day C.P. Semin. Diag. Pathol. 1998; 15: 246-258PubMed Google Scholar). It is possible that the presence of lipid droplets with larger diameters does not reflect their size in vivo and is an artifact of the fixation process.Formation of lipid droplets is considered to occur by a budding process at the ER (1Murphy D.J. Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google Scholar, 40Brown D.A. Curr. Biol. 2001; 11: R446-R449Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). From live cell studies, our results reveal that, following their biogenesis, droplets continue to attach to the ER membrane. As lipid droplets are a reservoir, their close association with the ER may be important for maintenance and expansion of the organelle during the cell cycle. Such an association also allows ready access to a storage reservoir under conditions where excess lipid is either synthesized or absorbed by cells. Thus, lipid droplets may be important to maintain lipid homeostasis and permit normal function of the ER in abnormal situations where lipid is either limiting or in excess. In HuH-7 cells, we observed that a proportion of lipid droplets had movements that were consistent with transient attachment to the microtubule network. Such trafficking was clearly distinct from the motion of droplets that were ER-associated. We presume that brief association of lipid droplets with microtubules indicates movement from one site on the ER to a second site. However, the factors that control lipid droplet association with microtubules are not known. We did not observe similar rapid movement in Vero cells. This cell type contains considerably fewer lipid droplets compared with HuH-7 cells and, because the proportion of lipid droplets that move rapidly in HuH-7 cells is low, it is possible that observations over longer time periods are necessary to identify microtubule-associated movement of lipid droplets in Vero cells. Alternatively, microtubule-related trafficking could be cell type-dependent. As well as acting as a source of lipid for membranes, lipid droplets have specialized functions in certain cell types, which may affect their interactions with cellular processes. For example, milk fat globules released by mammary epithelial cells are derived from lipid droplets secreted from the apical surface and it is proposed that movement of droplets to sites of secretion involve trafficking along microtubules (41Wu C.C. Howell K.E. Neville M.C. Yates J.R. McManaman J.L. Electrophoresis. 2000; 21: 3470-3482Crossref PubMed Scopus (188) Google Scholar). In addition, hepatocytes are a primary source for the production of very low density lipoprotein, and triglycerides stored in lipid droplets provide much of the lipid content in these particles (5Gibbons G.F. Islam K. Pease R.J. Biochim. Biophys. Acta. 2000; 1483: 37-57Crossref PubMed Scopus (248) Google Scholar). Mechanistically, this process is poorly understood but it may require mobilization of lipid droplets to regions of the ER where lipoprotein assembly occurs. Hepatocytes are the progenitor cells for the human hepatoma from which HuH-7 cells are derived and thus may maintain characteristics for the very low density lipoprotein assembly pathway that involve lipid droplets. Therefore, the association of lipid droplets with microtubules in HuH-7 cells may not be found more generally in other cell types.In summary, we have identified sequences within ADRP that are important for association with lipid droplets and have demonstrated that the protein can be utilized to examine the properties of these organelles. This has allowed analysis of the interaction between lipid droplets and other cellular processes. From our work and other studies where association with mitochondria, intermediate filaments (6Heid H.W. Moll R. Schwetlick I. Rackwitz H.R. Keenan T.W. Cell Tissue Res. 1998; 294: 309-321Crossref PubMed Scopus (356) Google Scholar, 42Francke W.W. Hergt M. Grund C. Cell. 1987; 49: 131-141Abstract Full Text PDF PubMed Scopus (205) Google Scholar), and peroxisomes (43Schrader M. J. Histochem. Cytochem. 2001; 49: 1421-1429Crossref PubMed Scopus (65) Google Scholar) have been observed, it appears that lipid droplets are not inert sites of lipid storage but actively interconnect with other organelles. Further studies on the consequences of these interactions will help to elucidate the full extent of the contribution of lipid droplets to cell metabolism. In mammalian cells, lipid droplets serve as storage organelles, consisting primarily of cholesterol ester and triacylglyerols (reviewed in Ref. 1Murphy D.J. Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google Scholar). Although long considered to be inert structures, there is now increasing evidence that they play active and diverse roles in the life cycle of cells. Recently, they have been implicated in maintenance of intracellular cholesterol balance and transport of lipids through association with caveolin proteins (2Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Crossref PubMed Scopus (274) Google Scholar). Moreover, the lipid stored in droplets is utilized for specialized purposes in certain cell types. For example, steroid hormones in steroidogenic cells are produced from cholesterol ester stored in lipid droplets (3Freeman D.A. Ascoli M. J. Biol. Chem. 1982; 257: 14231-14238Abstract Full Text PDF PubMed Google Scholar); mammary epithelial cells release milk fat globules from their apical surface that are directly derived from aggregated lipid droplets (4Mather I.H. Keenan T.W. J. Mammary Gland Biol. Neoplasia. 1998; 3: 259-273Crossref PubMed Scopus (282) Google Scholar); lipid droplets appear to be the primary source of fatty acids that are converted into triacylglycerols and incorporated into very low density lipoprotein in hepatocytes (5Gibbons G.F. Islam K. Pease R.J. Biochim. Biophys. Acta. 2000; 1483: 37-57Crossref PubMed Scopus (248) Google Scholar). There is also a correlation between particular human diseases and accumulation of lipid droplets that suggests they are markers of pathological changes. Such diseases include atheroma, steatosis, obesity, and some cancers (6Heid H.W. Moll R. Schwetlick I. Rackwitz H.R. Keenan T.W. Cell Tissue Res. 1998; 294: 309-321Crossref PubMed Scopus (356) Google Scholar, 7Murphy D.J. Vance J. Trends Biol. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). More recently, it has been suggested that aberrant targeting of the Nir2 protein to lipid droplets may induce changes to lipid transport that could correlate with retinal degeneration seen inDrosophila mutants (8Litvak V. Shaul Y.D. Shulewitz M. Amarilio R. Carmon S. Lev S. Curr. Biol. 2002; 12: 1513-1518Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Indeed, lipid droplets may have important functions in viral and parasitic infections (9Barba G. Harper F. Harada T. Kohara M. Goulinet S. Matsuura Y. Eder G. Schaff Zs. Chapman M.J. Miyamura T. Brechot C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1200-1205Crossref PubMed Scopus (573) Google Scholar, 10Charron A.J. Sibley L.D. J. Cell Sci. 2002; 115: 3049-3059Crossref PubMed Google Scholar, 11Hope R.G. McLauchlan J. J. Gen. Virol. 2000; 81: 1913-1925Crossref PubMed Scopus (192) Google Scholar, 12Hope R.G. Murphy D.J. McLauchlan J. J. Biol. Chem. 2002; 277: 4261-4270Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13Moriya K. Yotsuyanagi H. Shintani Y. Fujie H. Ishibashi K. Matsuura Y. Miyamura T. Koike K. J. Gen. Virol. 1997; 78: 1527-1531Crossref PubMed Scopus (576) Google Scholar). The surface of lipid droplets has a proteinaceous layer that is thought to prevent fusion with any adjacent lipophilic surface. However, analysis of the properties of surface proteins has received only limited attention. The most widely characterized lipid droplet-associated proteins are the perilipins, a family of polypeptides generated by alternative splicing from a single copy gene (14Lu X.Y. Gruia-Gray J. Copeland N.G. Gilbert D.J. Jenkins N.A. Londos C. Kimmel A.R. Mamm. Genome. 2001; 12: 741-749Crossref PubMed Scopus (181) Google Scholar), and adipocyte differentiation-related protein (ADRP,1 also termed adipophilin) (6Heid H.W. Moll R. Schwetlick I. Rackwitz H.R. Keenan T.W. Cell Tissue Res. 1998; 294: 309-321Crossref PubMed Scopus (356) Google Scholar, 15Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar, 16Gao J. Ye H. Serrero G. J. Cell. Physiol. 2000; 182: 297-302Crossref PubMed Scopus (96) Google Scholar, 17Jiang H.P. Serrero G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7856-7860Crossref PubMe
Four genetic polymorphisms in the APC and MCC genes at chromosome 5q21 were analysed for loss of heterozygosity (LOH) in 97 primary squamous carcinomas and adenocarcinomas of the lung. LOH was identified in at least two polymorphic loci in 41 percent of informative cases. There was no significant difference in the frequency of LOH between squamous carcinomas and adenocarcinomas. Within the adenocarcinoma group, however, LOH appeared to be more common in tumours having a bronchial origin (5/9; 56 per cent) than in parenchymal adenocarcinoma (6/21; 29 per cent). All 32 tumours showing LOH at one or more polymorphic sites were examined for mutations in the mutation cluster region (MCR) of APC by single-strand conformational polymorphism (SSCP) analysis. Mutations were not detected in any of these cases. We therefore propose that it is likely that a tumour suppressor gene on 5q other than APC is involved in the pathogenesis of lung cancer.
Abstract The results of genotypic analysis of 29 cases of malignant lymphoma are reported and the application of this technique for differentiating between Hodgkin's disease (HD) and non‐Hodgkin's lymphoma (NHL) is evaluated. Five cases with a differential diagnosis which included HD and NHL were analysed. These results are compared with those obtained for six B‐cell NHLs, nine T‐cell NHLs, and nine cases of HD. This report suggests that gene rearrangement analysis is useful in some cases in which the differential diagnoses includes HD and NHL as the absence of gene rearrangements is more consistent with a diagnosis of HD than of NHL. Two monoclonal antibodies reactive with the variable region of the T‐cell receptor β‐chain and molecular probes to the relevant variable region genes were used to assist in the diagnosis of T‐cell lymphoma. This report confirms that genotypic analysis is useful diagnostically when the results are assessed in the context of the histopathological findings.
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