Hybrid myelomas producing antibodies against a human neuroblastoma antigen present on fetal brain.

Spleen cells from mice immunized with a cultured human neuroblastoma were hybridized with the mouse plasmacytoma P3X63Ag8. Hybrid myelomas were screened for production of antibodies that reacted with human neuroblastomas but not with cells from other tissues. One of these hybridoma antibodies reacted with an antigen present on the six human neuroblastomas tested, one of two retinoblastomas, a glioblastoma, and fetal brain, but did not react with other tumors or tissues including adult human brain.

Genetic characteristics of the HeLa cell.

The genotype of the patient Henrietta Lacks from whose cervical carcinoma the HeLa cell was derived was deduced from the phenotypes of her husband and children, and from studies of the HeLa cell. Hemizygous expression of glucose-6-phosphate dehydrogenase in HeLa, together with the deduced heterozygosity of Mrs. Lacks, is consistent with clonal origin of her neoplasm.

Antisense inhibition of single copy N-myc expression results in decreased cell growth without reduction of c-myc protein in a neuroepithelioma cell line.

The N-myc gene is transiently expressed during normal embryonic development and abnormally expressed in several tumors of neuroendocrine origin. Little is known of the function of the N-myc gene product in either normal or neoplastic tissue. We utilized synthetic antisense oligodeoxynucleotides to specifically inhibit N-myc gene expression in the neuroepithelioma cell line CHP100. These cells contain single copy N-myc alleles but overexpress c-myc. N-myc antisense oligomer treatment was found to be growth inhibitory without affecting levels of c-myc protein. N-myc antisense oligomer-treated cells also lost the characteristic cellular heterogeneity displayed by CHP100 in vitro.

Inhibition of N-myc expression and induction of apoptosis by iron chelation in human neuroblastoma cells.

Neuroblastoma is the second most common solid malignancy of childhood. Enhanced expression of the amplified N-myc gene in the tumor cells may be associated with poor patient prognosis and may contribute to tumor development and progression. The use of deferoxamine mesylate (DFO), an iron chelator, to treat neuroblastoma is being investigated in national clinical studies. We show here by TUNEL assay and DNA laddering that DFO induces apoptosis in cultured human neuroblastoma cells, which is preceded by a decrease in the expression of N-myc and the altered expression of some other oncogenes (up-regulating c-fos and down-regulating c-myb) but not housekeeping genes. The decrease in N-myc expression is iron-specific but does not result from inhibition of ribonucleotide reductase, because specific inhibition of this iron-containing enzyme by hydroxyurea does not affect N-myc protein levels. Nuclear run-on and transient reporter gene expression experiments show that the decrease in N-myc expression occurs at the level of initiation of transcription and by inhibiting N-myc promoter activity. Comparison across neuroblastoma cell lines of the amount of residual cellular N-myc protein with the extent of apoptosis measured as pan-caspase activity after 48 h of iron chelation reveals no correlation, suggesting that the decrease in N-myc expression is unlikely to mediate apoptosis. In conclusion, chelation of cellular iron by DFO may alter the expression of multiple genes affecting the malignant phenotype by multiple pathways. Given the clinical importance of N-myc overexpression in neuroblastoma malignancy, decreasing N-myc expression by DFO might be useful as an adjunct to current

Molecular genetic characterization of epitope-specific monoclonal antibodies against the myc family proteins.

The myc family proteins were used to produce monoclonal antibodies with defined specificities. The pattern of mosaic homology among the myc family proteins facilitated the efficient identification of monoclonal antibodies specific to myc homology box sequences. Sequential epitopes for pan-myc reactive monoclonal antibodies produced against N-myc/c-myc fusion protein were further defined by use of truncated myc proteins made in E. coli and synthetic oligopeptides corresponding to myc box sequences. One class of antibodies was found to be specific to the first myc box sequence, whereas the other was found to be reactive with the third myc box sequence. Further development of anti-myc monoclonal antibodies, especially those antibodies specific to each myc box sequence, would be likely to facilitate analysis of the possible biological functions of the myc proteins in vivo and in vitro.

Prolonged N-myc protein half-life in a neuroblastoma cell line lacking N-myc amplification.

Genomic amplification of the oncogene N-myc is associated with rapid tumor progression and poor prognosis in patients with neuroblastoma (NB). However, 40% of NBs which lack N-myc amplification are also clinically aggressive. Factors other than N-myc copy number must therefore play a role in determining tumor progression in these NBs. We have established an unusual human NB cell line (NBL-S) from the primary tumor of a patient with rapidly progressive disease which lacks N-myc amplification. The doubling time in vitro (48 h) and the time from injection of 2 x 10(7) cells to detectable tumors in nude mice (46 days) in similar to NB cell lines with amplified N-myc. However, karyotype analysis reveals no evidence of double minutes (DMs), homogeneously staining regions (HSRs), or chromosome 1p deletions, features commonly seen in NB cell lines. The cells have the cell surface phenotype typical of N-myc amplified NB (HLA-A,B,C negative and HSAN 1.2 positive), and similar to other NB cell lines, N-myc RNA and protein are expressed. Interestingly, the half-life of the N-myc protein in NBL-S is prolonged (approximately 100 min) compared to the short N-myc protein half-life previously described in N-myc amplified NB cell lines (approximately 30 min). Because N-myc protein is thought to have a regulatory role, prolongation of the half-life of this protein may be an important factor in the regulation of growth in NBs which lack N-myc amplification and rapidly progress.

Glutaraldehyde fixation of the primary antibody-antigen complex on nitrocellulose paper increases the overall sensitivity of immunoblot assay.

A simple method to increase the overall sensitivity of immunoblot assays is described. The method is based on fixation of the primary monoclonal antibody-antigen complex on nitrocellulose paper by a divalent crosslinker, glutaraldehyde. The fixation of the antibody-antigen complex significantly increases the sensitivity of the assay. Unexpectedly, the glutaraldehyde treatment also improves the signal/noise ratio by decreasing non-specific binding of secondary antibodies and/or tertiary reagents. Thus, a significant overall improvement of immunoblot assays is achieved by this method.

The use of conventional antisera in the production of specific monoclonal antibodies.

Monoclonal antibodies specific for human alpha-2-macroglobulin (alpha 2M), a plasma glycoprotein, have been produced using a novel immunization method. Commercially available antisera to human alpha 2M was used to precipitate the antigen from whole serum. Immunization of animals with this immunoprecipitate resulted in the production of hybridomas with a specificity for human alpha-2-macroglobulin as confirmed by immunoprecipitation.

Detection of E. coli colonies expressing the v-sis oncogene product with monoclonal antibodies made against synthetic peptides.

A method for immunodetection of individual epitopes on eukaryotic proteins synthesized in E. coli colonies is described. The system is developed using monoclonal antibodies produced against the Ha-ras protein produced in E. coli JM103. Monoclonal antibodies made against a synthetic peptide from the v-sis oncogene sequence are then used to identify bacterial colonies in which the v-sis protein is being produced. Production of the v-sis protein by these E. coli colonies was confirmed by immunoblot analysis. The assay utilizes peroxidase conjugated anti-mouse immunoglobulin and 4-chloro-1-naphthol to detect the positive colonies and can detect on the order of 200 pg antigen per E. coli colony.

The use of conventional antisera in the production of specific monoclonal antibodies.

We have shown here that conventionally produced antisera aids in the production of specific monoclonal antibodies. The complex of immunoglobulin plus antigen acted as an effective immunogen in both rats and mice. Stable cell lines producing anit-human alpha 2M monoclonal antibodies to numerous antigenic sites on the alpha 2M molecule have been generated by this method. This method has also been used to produce monoclonal antibodies to human complement components. This procedure of immunizing mice with an immunoprecipitated product derived from conventionally produced antisera is a unique approach in that the antigen is conveniently enriched without tedious and time-consuming biochemical purification. In addition, the use of the immunoprecipitated product in conjunction with the immunoprecipitating antisera allows for rapid screening of the hybridomas in the initial stages when cell growth and maintenance is critical. This method thus further simplifies the task of obtaining a specific monoclonal antibody from a complex mixture. A final consideration is the wide availability of conventionally produced antisera to numerous proteins and other biological substances that could be used in the production of monoclonal antibodies, and consequently these antibodies could be used in more detailed studies of these same molecules.

Simple and rapid purification of monoclonal antibodies from cell culture supernatants and ascites fluids by hydroxylapatite chromatography on analytical and preparative scales.

A simple and rapid method for the purification of murine and human monoclonal antibodies from ascites fluids and cell culture supernatants is described. The method, based on the use of hydroxylapatite (HAP) column chromatography, is applicable on both analytical and preparative scales. In our work on purification of monoclonal antibodies, we have found that the combination of a single step elution of impurities followed by linear gradient elution of antibody provides an excellent purification of the antibody from cell culture and ascites fluids. The procedure provides very good resolution at high flow rates. The cell culture supernatant can be pumped on the preparative column at the rate of 2-3 ml/min without any measureable back pressure. The binding is independent of the flow rate. This method has been successfully used to purify several monoclonal antibodies of different subtypes from cell culture supernatants.

Production of continuous mouse plasma cell lines by transfection with human leukemia DNA.

Spleen cells from mice immunized with human cells were transfected with DNA from the human leukemia cell line, Reh. A calcium phosphate-DNA coprecipitate was introduced into the stimulated spleen cells by treatment with a polyethylene glycol-DMSO mixture. The cells which grew out from the transfected population could be passaged continuously in culture and cloned in semisolid agarose. The cell lines contain 40 acrocentric chromosomes, and Southern blot analysis with the cloned human Alu sequence indicates that human DNA is present. The transfected cell lines exhibit markers expressed on plasmacytoma cells and produce immunoglobulin in amounts equivalent to those produced by plasmacytoma cell lines. Five of nine cell lines tested produce antibodies that react with the human cells used to immunize the mice. These cell lines have been in culture for more than a year, and one of the lines has maintained a diploid karyotype and production of the specific antibody even after being passaged through a BALB/c mouse. Preliminary experiments indicate that these cells may be a useful model system for analysis of the early proliferative phase of leukocyte transformation.

A method of detecting neuroblastoma in human bone marrow by means of two monoclonal antibodies PI 153/3 and KE2.

A system is described to detect neuroblastoma (NBL) tumor cells in human bone marrow. The technique exploits the findings that NBL cells have little or no HLA antigen on the surface. Two monoclonal antibodies are used, PI153/3, IgM class, recognizes NBL and some pre-B lymphocytes and KE2 IgG class recognizes HLA. Two second antibodies are used, rhodamine-labeled anti-IgM and fluorescein-labeled anti-IgG. By means of fluorescence microscopy the neuroblastoma cells are labeled with rhodamine only, and the false + pre-B lymphocytes are double labeled with both rhodamine (Rh) and fluorescein (FI) since they are HLA+ and react with KE2. This method has been used to screen the marrow of 24 patients on 40 occasions and 64 laboratory preparations. It is possible to detect NBL cells at a concentration of 1:1000 marrow cells. The advantage of the technique is the fact that false positive cells can be defined because they have HLA surface antigen which neuroblastoma cells do not express.

Detection of neuroblastoma cells in human bone marrow using a combination of monoclonal antibodies.

Two different types of monoclonal antibodies, antineuroblastoma (PI153/3), and antilymphocyte (P3B1-C3) were used to identify and classify tumor cells in the bone marrow of patients with neuroblastoma and with other types of cancer. Cells expressing the antigens were detected with peroxidase-coupled anti-Ig. The cell-surface labeling is manifested as a dense black precipitate at the membrane visualized by light microscopy. The combination of the two antibodies gives specific staining patterns for each cell type. PI153/3+, P3B1-C3- is specifically associated with neuroblastoma cells. PI153/3+,P3B1-C3+ is expressed on blast cells from some types of acute lymphoblastic leukemia and a small subpopulation of normal lymphocytes. These monoclonal antibodies thus allow specific visual detection of single neuroblastoma cells in bone marrow samples. The results demonstrate how combinations of monoclonal antibodies can be effectively used to identify specific cell types by their expression of and lack of specific marker determinants. Application of this principle is particularly relevant for dissecting populations of related cells and/or molecules.

Hybridomas: a new dimension in biological analyses.

Since the first report of hybridomas producing monoclonal antibodies by Kohler and Milstein in 1975, this technique has spread to nearly all areas of biological, biochemical, and biomedical research. Watching the use of these methods spread from immunologists to cell biologists, developmental biologists, biochemists and to other biological disciplines and observing the nearly logarithmic increase in publications using these reagents has been in itself fascinating and informative. An overview of the development of this technology and its applications is presented including the use of monoclonal antibodies to study cell surface molecules, differentiation antigens, receptors, and histocompatibility antigens. The use of these antibodies to analyze microorganisms and parasitic antigens as well as their use in the genetic analysis of human cell surface antigens and the detection of polymorphic variation in enzymes and other proteins is discussed. Examples of the application of monoclonal reagents to the study of tumor cell biology including the labeling of metastatic tumor cells and the detection of cell surface molecules implicated in the regulation of growth control and cell division are provided.