CDR2L Antibodies: A New Player in Paraneoplastic Cerebellar Degeneration

Objective

Yo antibodies are associated with paraneoplastic cerebellar degeneration (PCD). We have characterized Yo sera by measuring CDR2 and CDR2L antibodies and the localization of their antigens.

Methods

Forty-two Yo sera from patients with paraneoplastic neurological syndromes (PNS), 179 sera from ovarian and 114 sera from breast cancer patients without PNS and 100 blood donors were screened for CDR2 and CDR2L antibodies by radioactive immune assay (RIA). Fluorescence microscopy was also used to determine the presence of CDR2 or CDR2L antibodies by staining of HeLa cells transfected with CDR2 or CDR2L fused to green fluorescent protein (GFP). Confocal microscopy was further used to localize the CDR2 and CDR2L proteins.

Results

RIA showed that 36 of the 42 Yo positive sera contained CDR2 and CDR2L antibodies whereas 6 sera contained only CDR2 antibodies. Five of the ovarian cancer patients had CDR2L antibodies and 4 of the breast cancer patients had either CDR2 or CDR2L antibodies. Only patients with both antibodies had PCD. RIA and staining of transfected cells showed similar results. Yo antibodies were not present in the 100 blood donors. Confocal microscopy showed that CDR2 and CDR2L were localized to the cytoplasm, whereas CDR2L was also present on the cell membrane.

Interpretation

Yo sera usually contain CDR2 and CDR2L antibodies and both antibodies are associated with PCD. Since only CDR2L is localized to the cell membrane it is likely that CDR2L antibodies may be of primary pathogenic importance for the development of PCD.     

Onconeural antibodies in sera from patients with various types of tumours

Purpose  We assessed the frequency and levels of onconeural antibodies in 974 patients with various types of tumours, but without apparent paraneoplastic neurological syndromes (PNS). Patients and methods  We included patients with the following tumours: 200 small-cell lung cancer (SCLC) patients, 253 breast cancer patients, 182 ovarian cancer patients, 266 uterine cancer patients and 73 thymoma patients, as well as 52 patients with PNS and cancer and 300 healthy blood donors. Sera were screened for amphiphysin, CRMP5, Hu, Ma2, Ri and Yo antibodies using a multi-well immunoprecipitation technique. Results  The most frequently detected antibodies were Hu followed by CRMP5. Ma2, Yo, amphiphysin and Ri antibodies were less common, but each was found at similar frequencies. Onconeural antibodies were present at similar levels in sera from the PNS control group and from cancer patients. Hu antibodies were rare in cancers other than SCLC. CRMP5 was the only antibody found in patients with thymoma and this antibody was more common among patients with thymoma than in other tumour patients. With one exception, coexisting antibodies were only found in patients with SCLC. The presence of onconeural antibodies in SCLC patients was not associated with prolonged survival. Conclusion  Onconeural antibodies are associated with various types of tumours suggesting that all antibodies should be included in the serological screening for possible PNS. The levels of onconeural antibody are not sufficiently sensitive to discriminate between cancer patients with PNS and those without.     

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.     

Antibody Repertoire in Paraneoplastic Cerebellar Degeneration and Small Cell Lung Cancer

The goal of this study is to determine whether patients with paraneoplastic cerebellar degeneration (PCD) and small-cell lung cancer (SCLC) have a specific repertoire of antibodies, if SOX1 antibodies (SOX1-ab) can predict the presence of SCLC, and if antibodies to cell surface antigens occur in this syndrome. Antibody analysis was done using immunohistochemistry on rat brain, immunoblot with recombinant antigens, screening of cDNA expression libraries, and immunolabeling of live neurons in 39 patients with PCD and SCLC. VGCC-ab were measured by RIA, and SOX1-ab, Hu-ab, and ZIC4-ab by immunoblot. Lambert-Eaton myastenic syndrome (LEMS) was present in 10 of 23 patients with electrophysiological studies. At least one antibody was detected in 72% of patients. The individual frequencies were: 49% SOX1-ab, 44% VGCC-ab, 31% Hu-ab, and 13% ZIC4-ab. SOX1-ab occurred in 76% of patients with VGCC-ab and 27% of those without VGCC-ab (p = 0.0036). SOX1-ab were not found in 39 patients with sporadic late-onset cerebellar ataxia, 23 with cerebellar ataxia and glutamic acid decarboxylase antibodies, and 73 with PCD and cancer types other than SCLC (31 without onconeural antibodies, 25 with Yo-ab , 17 with Tr-ab). Five patients (13%) had antibodies against unknown neuronal cell surface antigens but none of them improved with immunotherapy. One serum immunoreacted against the axon initial segment of neurons and another serum against ELKS1, a protein highly expressed in the cerebellum that interacts with the beta4-subunit of the VGCC. In conclusion, 72% of patients with PCD and SCLC had one or more antibodies that indicate the presence of this tumor. In these patients, VGCC-ab and SOX1-ab occur tightly associated. SOX1-ab are predictors of SCLC in ataxia patients with a specificity of 100% and sensitivity of 49%. Unlike limbic encephalitis with SCLC, antibodies to cell surface antigens other than VGCC-ab, are infrequent and do not predict response to treatment.     

Antibody to CCDC104 is associated with a paraneoplastic antibody to CDR2 (anti-Yo)

Patients with cancer may develop paraneoplastic neurological syndromes (PNS) in which onconeural antibodies are important diagnostic findings. As the functional role of onconeural antibodies is largely unknown, insight gained by identifying associated antibodies may help to clarify the pathogenesis of the PNS. In this study, we identified patients with Yo antibodies who also had antibodies to an uncharacterized protein called coiled-coil domain-containing protein 104 (CCDC104). We found a significant association between CCDC104 and Yo antibodies (4 of 38, 10.5%), but not other onconeural antibodies (0 of 158) (P = 0.007, Fisher’s exact test). The prevalence of CCDC104 antibodies was approximately similar in patients with cancer (8 of 756, 1.1%) and in healthy blood donors (2 of 300, 0.7%). CCDC104 antibodies were not associated with PNS, as this was found in only two of the ten CCDC104-positive patients. The CCDC104 protein, whose function is unknown, is expressed in various human tissues, including the brain, and is localized mainly to the nucleus, but is also found in the cytoplasm. The association between Yo and CCDC104 antibodies may indicate functional similarities.     

Developmental and Regional Changes of mRNA for a Cerebellar Protein (Spot 35) in the Rat Brain

Spot 35 protein is a cerebellar Ca-binding protein. Because Southern blot analysis showed evidence for the nucleotide sequence of spot 35 protein cDNA in the rat genome, we applied cDNA to quantitate the mRNA of rat spot 35 protein. The size of this mRNA was about 1,900 nucleotides in length, and that of mRNA from bovine cerebellum was larger. We also examined the developmental changes and regional distribution of spot 35 protein mRNA in rat brains by dot-blot analysis using cDNA as a probe. During postnatal days 1-20, a rapid increase of mRNA levels was observed. Further, the level of mRNA for spot 35 protein was found in the cerebellum and was negligible in the cerebral cortex, striatum, and hippocampus.     

Avidity of onconeural antibodies is of clinical relevance

Onconeural antibodies are important in the detection of paraneoplastic neurological syndromes (PNS). The avidity of Hu, Yo, and CRMP5 antibodies from 100 patients was determined by immunoprecipitation (IP), and 13 of the Yo positive sera were also tested by surface plasmon resonance (SPR). There was a significant association between the results from IP and SPR. Yo antibodies had higher avidity than Hu and CRMP5 antibodies, and both high- and low-avidity antibodies were associated with tumors and PNS. High-avidity Yo antibodies were mainly associated with ovarian cancer, whereas high-avidity Hu and CRMP5 antibodies were mainly associated with small-cell lung cancer. Low-avidity CRMP5 and Yo antibodies were less often detected by a commercial line blot than high-avidity antibodies. The failure to detect low-avidity onconeural antibodies may result in under diagnosis of PNS.     

Prostatic spermine-binding protein. Cloning and nucleotide sequence of cDNA, amino acid sequence, and androgenic control of mRNA level

cDNA for mRNA of an androgen-dependent spermine-binding protein (SBP) of rat ventral prostate was cloned by inserting cDNA into a dG-tailed expression vector, pUC8, and screening the expression library with anti-SBP antibodies. Hybrid-selected translation using plasmid DNA from positive clones yielded a 34-kDa protein which was immunoprecipitated by affinity-purified anti-SBP antibodies. SBP mRNA is about 1260 bases long as measured by Northern blot hybridization. An amino acid sequence deduced from the nucleotide sequence of the cDNA was identical to an amino acid sequence found in SBP. SBP is extremely rich in acidic residues. Aspartic and glutamic acids, which make up about 33% of the total sequence, comprise 89 of a stretch of 126 amino acids at the carboxyl-terminal end. By dot hybridization analysis, SBP mRNA was not detected in rat liver, kidney, brain, submaxillary gland, or uterus. The prostate levels of SBP mRNA were measured by mRNA translation and dot hybridization. SBP mRNA level decreased to less than 20% of normal 2 days after castration of rats, and this decrease was reversed by 5 alpha-dihydrotestosterone injection into castrated rats.     

The multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer

Although glyceraldehyde-3-phosphate dehydrogenase's (GAPDH) predilection for AU-rich elements has long been known, the expected connection between GAPDH and control of mRNA stability has never been made. Recently, we described GAPDH binding the AU-rich terminal 144 nt of the colony-stimulating factor-1 (CSF-1) 3' untranslated region (UTR), which we showed to be an mRNA decay element in ovarian cancer cells. CSF-1 is strongly correlated with the poor prognosis of patients with ovarian cancer. We investigated the functional significance of GAPDH's association with CSF-1 mRNA and found that GAPDH small interfering RNA reduces both CSF-1 mRNA and protein levels by destabilizing CSF-1 mRNA. CSF-1 mRNA half-lives were decreased by 50% in the presence of GAPDH small interfering RNA. RNA footprinting analysis of the 144 nt CSF-1 sequence revealed that GAPDH associates with a large AU-rich-containing region. The effects of binding of GAPDH protein or ovarian extracts to mutations of the AU-rich regions within the footprint were consistent with this finding. In a tissue array containing 256 ovarian and fallopian tube cancer specimens, we found that GAPDH was regulated in these cancers, with almost 50% of specimens having no GAPDH staining. Furthermore, we found that low GAPDH staining was associated with a low CSF-1 score (P = 0.008). In summary, GAPDH, a multifunctional protein, now adds regulation of mRNA stability to its repertoire. We are the first to evaluate the clinical role of GAPDH protein in cancer. In ovarian cancers, we show that GAPDH expression is regulated, and we now recognize that one of the many functions of GAPDH is to promote mRNA stability of CSF-1, an important cytokine in tumor progression.     

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.     

A novel protease homolog differentially expressed in breast and ovarian cancer.

BACKGROUND: Using differential display (DD), we discovered a new member of the serine protease family of protein-cleaving enzymes, named protease M. The gene is most closely related by sequence to the kallikreins, to prostate-specific antigen (PSA), and to trypsin. The diagnostic use of PSA in prostate cancer suggested that a related molecule might be a predictor for breast or ovarian cancer. This, in turn, led to studies designed to characterize the protein and to screen for its expression in cancer. MATERIALS AND METHODS: The isolation of protease M by DD, the cloning and sequencing of the cDNA, and the comparison of the predicted protein structure with related proteins are described, as are methods to produce recombinant proteins and polyclonal antibody preparations. Protease M expression was examined in mammary, prostate, and ovarian cancer, as well as normal, cells and tissues. Stable transfectants expressing the protease M gene were produced in mammary carcinoma cells. RESULTS: Protease M was localized by fluorescent in situ hybridization analysis to chromosome 19q13.3, in a region to which other kallikreins and PSA also map. The gene is expressed in the primary mammary carcinoma lines tested but not in the corresponding cell lines of metastatic origin. It is strongly expressed in ovarian cancer tissues and cell lines. The enzyme activity could not be established, because of difficulties in producing sufficient recombinant protein, a common problem with proteases. Transfectants were selected that overexpress the mRNA, but the protein levels remained very low. CONCLUSIONS: Protease M expression (mRNA) may be a useful marker in the detection of primary mammary carcinomas, as well as primary ovarian cancers. Other medical applications are also likely, based on sequence relatedness to trypsin and PSA.     

Isolation of a cDNA encoding a growth-arrest associated gene and characterization of its regulation

We are interested in understanding the molecular events associated with the growth-arrest of vascular SMCs. We constructed a subtracted cDNA library enriched in nucleotide sequences associated with quiescent SMCs. This library was screened with similarly subtracted ³²P-labeled cDNAs to identify growth-arrest associated cDNA clones. Characterization of 19 of these cDNA clones revealed that 9 hybridized to mRNAs that exhibited a 2–3 fold increase in growth-arrested SMCs. In addition, two other cDNAs hybridized to a 5 Kb mRNA that was elevated approximately 10-fold in high density growth-arrested SMCs. Genomic Southern blot hybridization and DNA sequencing analysis indicated that these cDNAs encoded the same gene (LG7) and that this gene may be a member of a multigene family or that it may contain a sequence shared by other unrelated genes. Augmented expression of LG7 was associated with both high cell density and serum deprivation induced growth-arrest. LG7 mRNA expression was down-regulated when SMCs were incubated with FBS or with reagents that arrest cells in early S-phase. Additional analysis with cell cycle specific inhibitors indicated that LG7 mRNA levels were also low when cells were blocked at the G₂ phase of the cell cycle but blockage at mitosis resulted in an elevated level of LG7 mRNA. We further demonstrated that the expression of LG7 was dependent on the presence of a relatively labile protein since protein synthesis inhibitors specifically blocked the expression of this mRNA but not the mRNA expression of α₁(III) collagen or ferritin H-chain. Finally, we demonstrated that Bt₂cAMP was able to induce mRNA expression of LG7 within 2 h, suggesting that this gene may be directly regulated via the cyclic-AMP-dependent protein kinase pathway.     

Identification and characterization of an ovary-selective isoform of epoxide hydrolase

A novel ovary-selective gene was identified by suppression subtractive hybridization (SSH) that is expressed only during the mouse periovulatory phase of a stimulated estrous cycle. Analysis of the protein encoded by the full-length cDNA revealed that the majority of it, with the exception of the first 44 amino acids, matched soluble epoxide hydrolase (Ephx2, referred to as Ephx2A). By comparing the cDNA sequence of this newly identified variant of soluble epoxide hydrolase (referred to as Ephx2B) with the mouse genome database, an exon was identified that corresponds to its unique 5' cDNA sequence. Through the use of an Ephx2A-specific probe, Northern blot analysis revealed that this mRNA was also expressed in the ovary, with the highest level of expression occurring during the luteal phase of a stimulated estrous cycle. In situ hybridization revealed that Ephx2B mRNA expression was restricted to granulosa cells of preovulatory follicles. Ephx2A mRNA expression, however, was detectable in follicles at different stages of development, as well as in the corpus luteum. Total ovarian epoxide hydrolase activity increased following the induction of follicular development, and remained elevated through the periovulatory and postovulatory stages of a stimulated estrous cycle. The change in enzyme activity paralleled the combined mRNA expression profiles for both Ephx2A and Ephx2B, thus supporting a role for epoxide metabolism in ovarian function.     

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N₂ fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.     

Comparative hybridization of an array of 21,500 ovarian cDNAs for the discovery of genes overexpressed in ovarian carcinomas

Comparative hybridization of cDNA arrays is a powerful tool for the measurement of differences in gene expression between two or more tissues. We optimized this technique and employed it to discover genes with potential for the diagnosis of ovarian cancer. This cancer is rarely identified in time for a good prognosis after diagnosis. An array of 21,500 unknown ovarian cDNAs was hybridized with labeled first-strand cDNA from 10 ovarian tumors and six normal tissues. One hundred and thirty-four clones are overexpressed in at least five of the 10 tumors. These cDNAs were sequenced and compared to public sequence databases. One of these, the gene HE4, was found to be expressed primarily in some ovarian cancers, and is thus a potential marker of ovarian carcinoma.