区别人小细胞肺癌和非小细胞肺癌的LSC系列单克隆抗体(简报)

人小细胞肺癌的发病率虽不及非小细胞肺癌,但其五年生存率却相对低得多,这两种细胞类型不仅在病理类型上表现不同,在许多其它性质上也表现不同。小细胞肺癌是一种异质性疾病,它由几种形态不同的瘤细胞组成,包括非小细胞肺癌在内;它易早期转移,能在肝、脑和骨髓中繁殖,具有L-Dopa胱羧酶,神经原特异的烯醇化酶(enolase),能合成神经递质和多肽激素蛙皮肽等特性。目前已有关于小细胞肺癌的单克隆抗体的报道,我们采用美国N I H建立的小细胞肺癌细胞株SH-77作免疫原,目前尚未见有关抗该细胞株的单克隆抗体的报道,现将我们制备的三个单抗体及其初步特异性鉴定结果简报如下     

McAb LC-1 against human lung cancer

Recently Ge et al reported LAC-122, LAC-210 and LAC-163 McAbs against Human non-small cell lung cancer and LSC McAbs against human lung small cell carcinoma. The immunotoxin, composed of McAb LAC-122 conjugated with Ricin A chain has been reported to have the significant cytotoxic effect in vitro on lung adenocarcinoma cell line SPC-A-1 by Tan et al. The LAC-122 alone has no effect on this target cell in the presence of complement from human, rabbit or guinea pig. The tumor associated antigens of human lung cancer have been recognized for many years, but only few reports deal with the common antigens or common epitopes of the lung cancer. From one fusion, 20 hybrids had been observed, all of these culture supernatants could react with target cell by IF. One of them after 4 th cloning, immunoglobulin isotype of the monoclonal antibody thus far obtained belonged to IgM and named to LC-1. Table 1 showed the results of ABC staining of LC-1 with a variety of tumors, normal adult and fetal tissues. From 12 non-small cell lung cancers, including 7 lung adenocarcinoma, 2 lung squamous carcinoma, 3 lung giant cell carcinoma, only one adenocarcinoma gave negative staining. As for 6 small cell lung cancers, all of them showed the positive reaction. It could be also reacted with 11 other tumors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human-human hybridomas secreting antibodies specific to human lung carcinoma

Summary Human Namalwa cells were screened in serum-free medium and in 6-thioguanine, then fused with human lymphocytes from lymph nodes of lung adenocarcinoma cancer patients. Extensive testing using 14 lung cancer cell lines, 11 other cancer cell lines and 4 normal fibroblast lines identified monoclonal antibodies produced by 4 hybridoma clones that reacted specifically with lung adenocarcinoma cells. These monoclonal antibodies also reacted with lung adenocarcinoma tissues and not normal tissues or erythrocytes of any blood type. These hybridoma clones grew and stably secreted the antibodies in serum-free medium as well as in serum-containing medium. Editor's Statement Identification of monoclonal antibodies that recognize human lung adenocarcinoma cells with reasonable specificity represents a potentially important development that may prove useful in diagnosis and therapy of neoplastic disease. The selection procedures and methods for propagation of the human-human hybridomas described in this paper also represent some novel approaches that may be of general application. David W. Barnes     

Systematic evaluation of microRNA processing patterns in tissues, cell lines, and tumors

Very little is known regarding regulation of microRNA (miRNA) biogenesis in normal tissues, tumors, and cell lines. Here, we profiled the expression of 225 precursor and mature miRNAs using real-time PCR and compared the expression levels to determine the processing patterns. RNA from 22 different human tissues, 37 human cancer cell lines, and 16 pancreas and liver tissues/tumors was profiled. The relationship between precursor and mature miRNA expression fell into the following four categories: (1) a direct correlation exists between the precursor and mature miRNA expression in all cells/tissues studied; (2) direct correlation of the precursor and mature miRNA exists, yet the expression is restricted to specific cell lines or tissues; (3) there is detectable expression of mature miRNA in certain cells and tissues while the precursor is expressed in all or most cells/tissues; or (4) both precursor and mature miRNA are not expressed. Pearson correlation between the precursor and mature miRNA expression was closer to one for the tissues but was closer to zero for the cell lines, suggesting that processing of precursor miRNAs is reduced in cancer cell lines. By using Northern blotting, we show that many of these miRNAs (e.g., miR-31, miR-105 and miR-128a) are processed to the precursor, but in situ hybridization analysis demonstrates that these miRNA precursors are retained in the nucleus. We provide a database of the levels of precursor and mature miRNA in a variety of cell types. Our data demonstrate that a large number of miRNAs are transcribed but are not processed to the mature miRNA.     

Induction of lymphomas on implantation of human oral squamous cell carcinomas in nude mice

Cancer cells from five oral cancer patients and pleomorphic adenoma cells from one individual were inoculated as single cell suspension into subcutis of 30 Swiss nude mice and tail vein of additional 30 mice. Further, tumor tissue pieces from three oral cancer patients were xenografted s.c. in 18 nude mice, and 10 mice were kept as controls. In animals implanted with tumor pieces, 7/18 (39%) mice, developed squamous cell carcinoma at the site of inoculation within 8-15 days, while tumors were not observed in mice inoculated with single cell suspension, up to 60/90 days. In 8/68 (12%) mice, white foci were observed in several tissues, with hepatomegaly and splenomegaly noted in 27/68 (39%) mice. Histopathological examination of various tissues revealed presence of large cell lymphoma in several organs in 14/68 (21%) mice. No regional or distant metastasis of the implanted oral tumor cells was detected. Mice injected with cells from pleomorphic adenoma, also demonstrated large cell lymphoma in 2/10 (20%) mice, whereas none of the 10 control animals showed any gross abnormalities or microscopic abnormalities in several organs. 2/16 (12%) lymphomas exhibited positive reaction with mouse B cell antibodies illustrating the murine origin of the lymphomas, and these were immunophenotyed as B cell lymphomas. The lymphomas were also examined with mouse T cell antibodies and none reacted positively with the mouse T cell antibodies. The lymphomas also failed to react with human T cell, B cell and human Leucocyte common antigen (LCA) antibodies, indicating that the induced lymphomas were not of human origin. The tumor specimens from seven of eight oral cancer patients and the pleomorphic adenoma patient induced lymphomas in nude mice. Thus it appears that xenografting oral tumor cells into nude mice may cause induction of the murine lymphomas, and this needs further investigation.     

Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer

Immunotherapy is emerging as a supplement to conventional cancer treatment, and identifying antigen targets for specific types of cancer is critical to optimizing therapeutic efficacy. Cancer/testis antigens are highly promising targets for immunotherapy due to their cancer-specific expression and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many other CT antigens, such as MAGE-A proteins. Surprisingly, we detected no ADAM2, CALR3 and SAGE1 in the 67 lung cancers (mainly non-small lung cancer) and 189 breast cancers, while MAGE-A proteins were present in 15% and 7–16% of these tumor types, respectively. Treatment with DNA methyltransferase inhibitors has been proposed as an attractive strategy to increase the expression of cancer/testis antigens in tumors before immunotargeting; however, neither ADAM2, CALR3 nor SAGE1 could be significantly induced in lung and breast cancer cell lines using this strategy. Our results suggest that ADAM2, CALR3 and SAGE1 cancer/testis antigens are not promising targets for immunotherapy of breast and lung cancer.     

A new small cell lung cancer (SCLC)-specific marker discovered through antigenic subtraction of neuroblastoma cells

Small cell lung cancer (SCLC) is an aggressive form of lung cancer associated with cigarette smoking and presently accounts for approximately 20% of all lung cancer cases. SCLC cells derive from a neuroendocrine origin and therefore their antigenic profile coincides, to a great extent, with that of neuroendocrine cells. Multiple attempts to generate SCLC-specific MoAbs during the past decade have failed because all SCLC-specific MoAbs isolated also react against neuroendocrine tissues or normal immune cells. Cross-reactivity with normal antigens raises safety concerns due to the inevitable toxicity of such interactions and the dreaded effects. The concept of DIAAD trade mark ( Differential Immunization for Antigen and Antibody Discovery) provides for an immune response that can be effectively focused on cancer antigens. The object is to overcome obstacles resulting from an antigenic hierarchical pattern biased towards a response to dominant antigens in order to induce a robust immune response to cancer antigens. Cancer antigens are weak or nonimmunogenic molecules. Due to the fact that the immune system responds more strongly to immunodominant antigens than to weak immunogenic antigens, cancer cell proliferation is unencumbered. DIAAD employs protocols of induction of tolerance and immunity, conducted in sequential order to "biologically subtract" the immune response of dominant antigens expressed by normal cells. This biological subtraction is achieved in a laboratory animal by first eliminating the immune response to the normal cells or closely related cancer cells, followed by immunization of the same laboratory animal with diseased cells. This procedure directs the immune response exclusively towards antigens expressed by the diseased and not the normal cells. Our objective was to use DIAAD to generate monoclonal antibodies specific to SCLC antigens that are not shared by neuroendocrine cells by contrasting a pool of human SCLC cell lines with a pool of human neuroendocrine cancer cell lines. Four monoclonal antibodies reacted strongly and exclusively with SCLC cells and identified a membrane molecule comprising a single chain glycoprotein. Two of four antibodies were selected for a detailed analysis that revealed a narrow tissue specificity of antigen expressed by colon, lung, and pancreatic cancers (less than 20% staining was found on breast, ovarian and prostate cancer). These antibodies did not bind to various other cancers such as kidney, carcinoid, lymphoma, sarcoma, adrenal, liver, melanoma, seminoma, leiomyoma, basal cell cancer, or undifferentiated cancer. The epitope recognized by the selected MoAbs was destroyed with the removal of carbohydrates from SCLC cells. This result does not exclude the possibility of protein-carbohydrate cooperation in epitope recognition. However, it strongly suggests the pivotal role of carbohydrates in antibody binding to this molecule. Upon binding to the extracellular molecule on SCLC cells, the antibodies were shown to internalize. A low or insignificant level of internalization was recorded following incubation of the antibodies with neuroendocrine-derived tumors. The capacity of these antibodies to internalize upon binding the extracellular receptors renders them potential candidates for prodrug or immunotoxin-targeted therapeutics. In a qualitative experiment involving immunoaffinity purification, the SCLC antigen was shown to be differentially detected in sera of SCLC patients. Plans are being generated to explore the possible utility of this novel SCLC-specific antigen recognized by the above MoAbs as a new biomarker for early diagnosis of the disease, as well as for therapeutic intervention for SCLC.     

肺癌组织和肿瘤细胞系中BRG1的表达分析

BRG1（brahma—related gene 1）是进化上高度保守的SWI／SNF染色质重塑复合物的成员之一．研究表明：BRG1具有抑瘤基因的特征,可能与肿瘤的发生发展有关．我们采用RT—PCR、Northern杂交和Western blotting证实：肺腺癌细胞系A549和鼻咽癌细胞系HNE2、HNE3、CNE1中无BRG1的表达,而肺鳞癌细胞系NCI-H520、永生化正常人支气管上皮细胞系HBE和鼻咽癌细胞系HONE1、HNE1、CNE2中有BRG1的表达．同时,通过RT—PCR检测10例肺癌组织标本．发现60％（6／10）的肺癌组织中日RGG1的mRNA水平明显下调,而配对正常肺组织中BRG1的mRNA表达未见改变．对29例肺癌组织和10例配对正常肺组织切片进行免疫组化染色,结果显示：肺癌组织中BRG1蛋白表达的阳性率为37．9％（11／29）,配对正常肺组织中BRG1蛋白表达的阳性率为90％（9／10）,两者的差异有显著性（P〈0．05）．这提示BRG1确实在肺癌组织及多种肿瘤细胞系中表达下调或缺失,在肺癌发病过程中可能起一定的作用．     

Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines

The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors.     

Epithelial tight junction proteins as potential antibody targets for pancarcinoma therapy

Recombinant monoclonal antibodies are beginning to revolutionize cancer therapy. In combination with standard chemotherapy, high response rates have been reported with antibodies of the human IgG1 isotype for treatment of non-Hodgkins lymphoma and breast cancer. It is becoming apparent that targets for antibody-based therapies do not necessarily need to be absent from normal tissues but can be present there either in low copy numbers or with binding epitopes shielded from the therapeutic antibody. Here, we studied whether claudin proteins that form tight junctions in normal epithelia are still expressed on carcinoma cells and whether their extracellular domains can be recognized by antibodies. We show that mRNAs of claudins 1, 3, 4, and 7 are all expressed in different human carcinoma cell lines, while claudin 8 was selectively expressed in breast and pancreas cancer lines. Chicken polyclonal antibodies were raised against peptides contained within predicted extracellular domains of claudins 1, 3, and 4. Affinity-purified IgG fractions for claudins 3 and 4 were monospecific and bound to human breast and colon carcinoma lines, but not to a line of monocytic origin. Claudin 3 antibodies also homogeneously stained human renal cell carcinoma tissue and micrometastatic tumor cells as identified by cytokeratin staining in bone marrow biopsies of breast cancer patients. Fluorescence-activated cell sorting and immunocytochemistry indicated that claudin antibodies bound to the surface of tumor cells. By analogy to other tumor-associated antigens that are differentially accessible to antibodies on tumor vs normal tissue, we propose that certain claudin proteins have potential as targets for novel antibody-based therapies of carcinomas.     

Screening and identification of novel B cell epitopes in human heparanase and their anti-invasion property for hepatocellular carcinoma

Background  The aim of this study was to screen and identify novel B cell epitopes within the human heparanase protein and to investigate the impact of self-developed anti-heparanase polypeptide antibodies on growth and invasion of HCCLM6 human hepatocellular carcinoma cells in vitro. Methods  The flexible regions of secondary structure and the B cell epitopes of the human heparanase amino acid sequence were predicted by DNAStar and Bcepred software.The multiple antigenic peptides (MAP) of the epitopes were synthesized in eight-branched form. Rabbits were immunized with the eight-branched MAPs mixed with the universal T-helper epitope human IL-1β peptide (VQGEESNDK, amino acid 163–171). The immunogenicity of the synthesized peptides was evaluated by ELISA, western blot and immunohistochemistry. The impact of the self-developed rabbit anti-heparanase polyclonal antibodies on growth and invasion ability of HCCMLM6 cells were analyzed in a cell culture model. The cells were first treated with one of the three antibodies, respectively, and then measured by using MTT, flow cytometry, plate clone formation, invasion assay and heparan sulfate degrading enzyme assay. Results  The three amino acid sequences 1–15 (MAP1), 279–293 (MAP2), and 175–189 (MAP3) in the large subunit of the human heparanase protein were predicted as its most potential epitopes. ELISA, western blot and immunohistochemistry analysis showed that all three MAPs were capable to induce high titer of serum antibodies. Antibodies induced by MAP1 and MAP2 were high specific. Furthermore, anti-MAP2 antibodies showed the strongest avidity towards liver cancer tissues. Under the treatment with the three anti-heparanase antibodies, respectively, the growth, cell cycle and clone formation of the cells remained unchanged when compared with a treatment with normal rabbit IgG. However, an inhibition of cell invasiveness and heparanase activity could be detected under the treatment with anti-MAP1- or anti-MAP2-antibody (with a terminal concentration of 100 μg/ml). The cell invasiveness was decreased by 54 and 38%, respectively, the heparanase activity by 43 and 39%, respectively. Conclusion  The multiple antigenic peptides MAP1 (AC 1–15) and MAP2 (AC 279–293) may be the dominant B cell epitopes in the human heparanase protein. The induced polypeptide antibodies can effectively inhibit the heparanase activity of HCCLM6 liver cancer cells and therefore influence their invasion ability, which provides a theoretic basis for the development of anti-heparanase antibodies and their clinical use as vaccine.     

Studies on the human tumor nucleolar antigens

With rabbit antibodies to nuclear 0.01 M Tris-HCl, pH 8, extract or "nucleolar preparations" of human HeLa S3 cells and fluorescein-labeled goat antirabbit antibodies, bright nucleolar immunofluorescence was observed in human adenocarcinomas, squamous cell carcinomas, sarcomas, hematological neoplasms, and other malignant tumors. With these antibodies, nucleolar immunofluorescence was not found in most normal tissue specimens, benign adenomas, hyperplastic tissues, and specimens of inflammatory diseases. A study was made on the presence in benign and malignant breast tumors of a common nucleolar antigen previously found in a broad range of human malignant tumors. Bright nucleolar immunofluorescence was observed in 19/20 (95%) of known breast cancer specimens. In the group of 80 unknown samples in the "blind" study, 75 (94%) were correctly identified as malignant or benign on the basis of the presence and distribution of the nucleolar fluorescence. In a group of 67 samples in which the nucleolar fluorescence was either readily observed or virtually absent, 47/48 (98%) of the malignant tumors were correctly identified. Of the bening lesions or normal breast specimens, 18/19 (95%) were correctly identified as negative for nucleolar fluorescence. These studies extend the results previously reported for a common nucleolar antigen in a broad range of human cancers to a larger series of malignancies of a particular organ. The tumor nucleolar antigen(s) were partially characterized by isoelectric focusing on 4% polyacrylamide gels. One major band had a pI of 6.3 and a minor band had a pI of 6.1. These antigens were not found in the normal human liver nucleoli.     

Methods for production of monoclonal antibodies with specificity for human lung cancer cells

We have developed a screening strategy and technology to produce monoclonal antibodies with specificity for human lung cancer cells. Mice and rats were immunized with well-characterized tissue culture lines of human small cell lung cancer (SCLC), mouse myeloma x spleen hybrids formed by the technique of Kohler and Milstein, and the resulting culture fluids were screened for antibody binding phenotype using a radioimmunoassay. To facilitate testing large numbers of culture fluids, a 96-well, microtiter based, reusable, replicating device was designed. Using this, many hybridoma culture fluids were replica plated for antibody binding tests on a series of human target cell plates. Hybrids producing antibodies that reacted with the immunizing SCLC line and another independent SCLC line, but not with autologous B-lymphoblastoid cells derived from one of the patients, were identified, selected, and then repeatedly recloned using the same screening strategy. With this technology, hybridomas representing less than 0.5% of all hybrids generated could be isolated and stable antibody producing cultures derived. Such antibodies reacted with a panel of well-characterized SCLC lines and SCLC samples taken directly from patients but not with a variety of normal tissues. Using these antibodies we can demonstrate: tumor cell contamination of bone marrow specimens, marked heterogeneity of antigen expression on cells within individual SCLC lines and individual patients, and inhibition of clonal growth of SCLC lines in soft agarose assays. All of these findings have potential clinical and cell biologic application.     

Survivin--a universal tumor antigen

Tumor-associated antigens recognized by cellular effectors of the immune system are potential targets for antigen-specific cancer immunotherapy. These antigens are classified as tissue (melanocyte)-specific proteins, cancer-testis antigens (proteins expressed in normal testis and various cancers), tumor-specific peptides derived from mutations in tumor cells, and others. Clinical studies with peptides and proteins derived from these antigens have been initiated to study the efficacy of inducing specific cytotoxic T lymphocytes (CTL) responses in vivo. However, most of the peptide epitopes used in these vaccination trials are melanocyte-specific, and these peptides cannot be applied for tumors of non-melanocyte origin. Furthermore, the expression of most tumor antigens is heterogeneous among tumors from different patients and can even vary among metastases obtained from one patient. Immune selection of antigen loss variants may prove to be an additional obstacle for the clinical applicability of most of the known CTL epitopes. Recently, a new tumor antigen, survivin, has been identified on the basis of spontaneous CTL responses in different cancer patients. Survivin is expressed in most human neoplasms, but not in normal, differentiated tissues. Importantly, downregulation or loss of survivin would severely inflict the growth potential of the tumor cell. Since survivin is expressed by a variety of different tumors MHC-restricted survivin epitopes may serve as important and widely applicable targets for anti-cancer immunotherapeutic strategies.     

Matrix-associated heparan sulfate proteoglycan: core protein-specific monoclonal antibodies decorate the pericellular matrix of connective tissue cells and the stromal side of basement membranes

Cultured human lung fibroblasts produce a large, nonhydrophobic heparan sulfate proteoglycan that accumulates in the extracellular matrix of the monolayer (Heremans, A., J. J. Cassiman, H. Van den Berghe, and G. David. 1988. J. Biol. Chem. 263: 4731-4739). A panel of four monoclonal antibodies, specific for four distinct epitopes on the 400-kD core protein of this extracellular matrix heparan sulfate proteoglycan, detects similar proteoglycans in human epithelial cell cultures. Immunohistochemistry of human tissues with the monoclonal antibodies reveals that these proteoglycans are concentrated at cell-matrix interfaces. Immunogold labeling of ultracryosections of human skin indicates that the proteoglycan epitopes are nonhomogeneously distributed over the width of the basement membrane. Immunochemical investigations and amino acid sequence analysis indicate that the proteoglycan from the fibroblast matrix shares several structural features with the large, low density heparan sulfate proteoglycan isolated from the Engelbreth-Holm-Swarm sarcoma. Thus, both epithelial cell sheets and individual mesenchymal cells accumulate a large heparan sulfate proteoglycan(s) at the interface with the interstitial matrix, where the proteoglycan may adopt a specific topological orientation with respect to this matrix.     

Very Long-Chain Acyl-CoA Synthetase 3: Overexpression and Growth Dependence in Lung Cancer

Lung cancer is the leading cause of cancer deaths worldwide. In the United States, only one in six lung cancer patients survives five years after diagnosis. These statistics may improve if new therapeutic targets are identified. We previously reported that an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), is overexpressed in malignant glioma, and that depleting glioblastoma cells of ACSVL3 diminishes their malignant properties. To determine whether ACSVL3 expression was also increased in lung cancer, we studied tumor histologic sections and lung cancer cell lines. Immunohistochemical analysis of normal human lung showed moderate ACSVL3 expression only in bronchial epithelial cells. In contrast, all of 69 different lung tumors tested, including adeno-, squamous cell, large cell, and small cell carcinomas, had robustly elevated ACSVL3 levels. Western blot analysis of lung cancer cell lines derived from these tumor types also had significantly increased ACSVL3 protein compared to normal bronchial epithelial cells. Decreasing the growth rate of lung cancer cell lines did not change ACSVL3 expression. However, knocking down ACSVL3 expression by RNA interference reduced cell growth rates in culture by 65–76%, and the ability of tumor cells to form colonies in soft agar suspension by 65–80%. We also conducted studies to gain a better understanding of the biochemical properties of human ACSVL3. ACSVL3 mRNA was detected in many human tissues, but the expression pattern differed somewhat from that of the mouse. The enzyme activated long- and very long-chain saturated fatty acid substrates, as well as long-chain mono- and polyunsaturated fatty acids to their respective coenzyme A derivatives. Endogenous human ACSVL3 protein was found in a punctate subcellular compartment that partially colocalized with mitochondria as determined by immunofluorescence microscopy and subcellular fractionation. From these studies, we conclude that ACSVL3 is a promising new therapeutic target in lung cancer.