Search

We used phage display to generate surrogate peptides that define the hotspots involved in protein-protein interaction between insulin and the insulin receptor. All of the peptides competed for insulin binding and had affinity constants in the high nanomolar to low micromolar range. Based on competition studies, peptides were grouped into non-overlapping Sites 1, 2, or 3. Some Site 1 peptides were able to activate the tyrosine kinase activity of the insulin receptor and act as agonists in the insulin-dependent fat cell assay, suggesting that Site 1 marks the hotspot involved in insulin-induced activation of the insulin receptor. On the other hand, Site 2 and 3 peptides were found to act as antagonists in the phosphorylation and fat cell assays. These data show that a peptide display can be used to define the molecular architecture of a receptor and to identify the critical regions required for biological activity in a site-directed manner.

Pancreatic cancer is an extremely aggressive neoplasm whose incidence equals its death rate. Despite intensive analysis, the genetic changes that mediate pancreatic cancer development and effective therapies for diminishing the morbidity associated with this disease remain unresolved. Through subtraction hybridization, we have identified a gene associated with induction of irreversible growth arrest, cancer reversion, and terminal differentiation in human melanoma cells, melanoma differentiation associated gene-7 ( mda- 7). Ectopic expression of mda- 7 when using a recombinant adenovirus, Ad. mda- 7, results in growth suppression and apoptosis in a broad spectrum of human cancers with diverse genetic defects, without exerting deleterious effects in normal human epithelial or fibroblast cells. Despite the apparently ubiquitous antitumor effects of mda- 7, pancreatic carcinoma cells are remarkably refractory to Ad. mda -7 induced growth suppression and apoptosis. In contrast, the combination of Ad. mda- 7 with antisense phosphorothioate oligonucleotides, which target the K-ras oncogene (a gene that is mutated in 85 to 95% of pancreatic carcinomas), induces a dramatic suppression in growth and a decrease in cell viability by induction of apoptosis. In mutant K- ras pancreatic carcinoma cells, programmed cell death correlates with expression and an increase, respectively, in MDA -7 and BAX proteins and increases in the ratio of BAX to BCL- 2 proteins. Moreover, transfection of mutant K- ras pancreatic carcinoma cells with an antisense K- ras expression vector and infection with Ad. mda- 7 inhibits colony formation in vitro and tumorigenesis in vivo in nude mice. These intriguing observations demonstrate that a combinatorial approach, consisting of a cancer-specific apoptosis-inducing gene and an oncogene inactivation strategy, may provide the foundation for developing an effective therapy for pancreatic cancer.

Cancer is a progressive disease culminating in acquisition of metastatic potential by a subset of evolving tumor cells. Generation of an adequate blood supply in tumors by production of new blood vessels, angiogenesis, is a defining element in this process. Although extensively investigated, the precise molecular events underlying tumor development, cancer progression, and angiogenesis remain unclear. Subtraction hybridization identified a genetic element, progression elevated gene-3 (PEG-3), whose expression directly correlates with cancer progression and acquisition of oncogenic potential by transformed rodent cells. We presently demonstrate that forced expression of PEG-3 in tumorigenic rodent cells, and in human cancer cells, increases their oncogenic potential in nude mice as reflected by a shorter tumor latency time and the production of larger tumors with increased vascularization. Moreover, inhibiting endogenous PEG-3 expression in progressed rodent cancer cells by stable expression of an antisense expression vector extinguishes the progressed cancer phenotype. Cancer aggressiveness of PEG-3 expressing rodent cells correlates directly with increased RNA transcription, elevated mRNA levels, and augmented secretion of vascular endothelial growth factor (VEGF). Furthermore, transient ectopic expression of PEG-3 transcriptionally activates VEGF in transformed rodent and human cancer cells. Taken together these data demonstrate that PEG-3 is a positive regulator of cancer aggressiveness, a process regulated by augmented VEGF production. These studies also support an association between expression of a single nontransforming cancer progression-inducing gene, PEG-3, and the processes of cancer aggressiveness and angiogenesis. In these contexts, PEG-3 may represent an important target molecule for developing cancer therapeutics and inhibitors of angiogenesis.

Several components of the eukaryotic protein synthesis apparatus have been associated with oncogenic transformation of cells. Altered expression of translation elongation factor 1 alpha (EF-1 alpha), a core component of protein synthesis and closely related sequences have been linked with transformed phenotypes by several independent studies, in diverse systems. A dominant acting oncogene, prostate tumor inducing gene-1 (PTI-1) has provided further evidence for this link. PTI-1 appears to be a hybrid molecule with components derived from both prokaryotic and eukaryotic origins. The predicted protein coding moiety represents an EF-1 alpha molecule, truncated N-terminal to amino acid residue 68 and having six additional point mutations. This coding sequence is fused to a 5' untranslated region (UTR) showing strongest homology to ribosomal RNA derived from Mycoplasma hyopneumoniae. Expression studies using the cloned cDNA in nude mouse tumor formation assays have confirmed the oncogenic nature of the molecule. A broad spectrum of tumor derived cell lines, from varied tissue sources and blood samples from patients having confirmed prostate carcinoma, all scored positive for expression of PTI-1, while corresponding normal tissues or blood samples were negative. Based on its near identity to EF-1 alpha, it is proposed that PTI-1 represents a new class of oncogene whose transforming capacity probably arises through mechanisms including: (i) protein translational infidelity, resulting in the synthesis of mutant polypeptides due to loss of proofreading function during peptide chain elongation, (ii) by its association with and alteration of the cytoskeleton, (iii) by impinging on one particular or several different signal transduction pathways through its properties as a G-protein.

A differentiation induction subtraction hybridization strategy is being used to identify and clone genes involved in growth control and terminal differentiation in human cancer cells. This scheme identified melanoma differentiation associated gene-7 ( mda- 7), whose expression is up-regulated as a consequence of terminal differentiation in human melanoma cells. Forced expression of mda- 7 is growth inhibitory toward diverse human tumor cells. The present studies elucidate the mechanism by which mda- 7 selectively suppresses the growth of human breast cancer cells and the consequence of ectopic expression of mda- 7 on human breast tumor formation in vivo in nude mice. Infection of wild-type, mutant, and null p53 human breast cancer cells with a recombinant type 5 adenovirus expressing mda- 7, Ad. mda- 7 S, inhibited growth and induced programmed cell death (apoptosis). Induction of apoptosis correlated with an increase in BAX protein, an established inducer of programmed cell death, and an increase in the ratio of BAX to BCL-2, an established inhibitor of apoptosis. Infection of breast carcinoma cells with Ad. mda- 7 S before injection into nude mice inhibited tumor development. In contrast, ectopic expression of mda- 7 did not significantly alter cell cycle kinetics, growth rate, or survival in normal human mammary epithelial cells. These data suggest that mda- 7 induces its selective anticancer properties in human breast carcinoma cells by promoting apoptosis that occurs independent of p53 status. On the basis of its selective anticancer inhibitory activity and its direct antitumor effects, mda- 7 may represent a new class of cancer suppressor genes that could prove useful for the targeted therapy of human cancer.

The importance of angiogenesis in malignant tumor growth has been interpreted mainly in terms of oxygen and nutrient supply. Here we demonstrate its fundamental role for tumor invasion of malignant human keratinocytes in surface transplants on nude mice. Distinct patterns of angiogenesis and vascular endothelial growth factor receptor-2 (VEGFR-2) expression allowed us to distinguish between benign and malignant cells. Functional inactivation of VEGF-R2 by a blocking antibody disrupted ongoing angiogenesis and prevented invasion of malignant cells, without reducing tumor cell proliferation. The reversion of a malignant into a benign phenotype by halting angiogenesis demonstrates a significant function of vascular endothelium for tumor invasion.

Antisense oligodeoxynucleotides offer potential as therapeutic agents to inhibit gene expression. Recent evidence indicates that oligodeoxynucleotides designed to target specific nucleic acid sequences can interact nonspecifically with proteins. This report describes the interactive capabilities of phosphorothioate oligodeoxynucleotides of defined sequence and length with two essential protein tyrosine receptors, flk-1 and epidermal growth factor receptor (EGFR), and their effects on receptor signaling in a transfected and tumor cell line, respectively. Phosphorothioate oligodeoxynucleotides bound to the cell surface, as demonstrated by fluorescence-activated cell-sorter analyses (FACS), and perturbed receptor activation in the presence and absence of cognate ligands, EGF (EGFR) and vascular endothelial growth factor (flk-1), in phosphorylation assays. Certain phosphorothioate oligodeoxynucleotides interacted relatively selectively with flk-1 and partially blocked the binding of specific anti-receptor monoclonal antibodies to target sites. They stimulated EGFR phosphorylation in the absence of EGF but antagonized ligand-mediated activation of EGFR and flk-1. In vivo studies showed that a nonspecific phosphorothioate oligodeoxynucleotide suppressed the growth of glioblastoma in a mouse model of tumorigenesis. These results emphasize the capacity of phosphorothioate oligodeoxynucleotides to interact with cells in a sequence-selective nonantisense manner, while associating with cellular membrane proteins in ways that can inhibit cellular metabolic activities.

For prostate cancer, a correlation exists between overexpression of the epidermal growth factor receptor (EGFR) and poor clinical prognosis. In addition, late-stage metastatic disease is characterized by a change from a paracrine to an autocrine mode of expression for TGF-alpha, the ligand for the EGFR. These observations suggest that activation of the EGFR may be important for the growth of prostatic carcinoma in situ, and blockade of the receptor-ligand interaction may offer a means of therapeutic intervention for this disease. We describe the biologic effects of a chimeric anti-EGFR monoclonal antibody, C225, on several human prostate tumor cell lines in culture and the tumor inhibitory properties of the antibody for the treatment of human prostate carcinoma xenografts in nude mice. In vitro analysis of the EGFR from androgen-responsive and independent prostatic carcinoma cell lines revealed that C225 blocked EGF-induced receptor activation and induced internalization of the receptor. In vivo, a treatment regimen of C225 alone or antibody plus doxorubicin significantly inhibited tumor progression of well-established DU145 and PC-3 xenografts in nude mice. These results suggest that C225 may have utility for the treatment of human prostate carcinoma in a clinical setting.

The flk-2 tyrosine kinase receptor is expressed on hematopoietic stem cells and on acute leukemias (AML and ALL). We have isolated a rat monoclonal antibody (71E1) that binds to this receptor with a relative affinity of 5 nM. The antibody immunoprecipitates both murine and human forms of flk-2 and can block receptor activation by its cognate ligand. In addition, 71E1 inhibits the in vitro proliferation of the murine leukemic cell line, M1, that expresses high levels of flk-2. These results suggest that 71E1 may have utility as both a reagent for elucidating the biological role of flk-2 in hematopoiesis and as an immunotherapeutic in the treatment of acute leukemias.

Small cell lung carcinoma (SCLC) represents about 25% of all lung cancers. Human SCLC shows neuroendocrine features such as the production of neural peptide hormones, marker enzymes and neurosecretory granules, and the expression of neural cell adhesion molecules (NCAMs). Although SCLC is sensitive to both chemotherapy and radiation, prognosis remains poor due to the appearance of post-treatment chemo- and radioresistant variants. Monoclonal antibodies (MAbs) have been developed that bind to SCLC tumor antigens. We have used similar technology to define another SCLC marker designated gP94/115. The MAb CR101 binds to a highly glycosylated, cell-surface antigen associated with SCLC. In vitro expression of the antigen appears to be restricted to cell lines of SCLC origin. Enzymatic removal of the sugars resolves the antigen into two proteins of 94 and 115 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fluorescence-activated cell sorting (FACS) analysis confirms the antibody's specificity. These results indicate that CR101 may recognize a novel protein expressed by SCLC.

The trpE (or anthranilate synthetase) gene product has been used extensively as a fusion protein for the expression of a myriad of biologically active proteins. A trpE construct can be produced in high yield, is relatively resistant to proteolysis, and separates from the bulk of E. coli proteins because of its insolubility. We have isolated and characterized a monoclonal antibody against the TrpE protein for use as a detection and immunoaffinity reagent. The MAb, TRP 7.4, is highly specific for the TrpE protein and has a relative affinity of 1.0 ng. The antibody can also be used to detect TrpE constructs on Western blots. In addition, TRP 7.4 has been used to purify a TrpE-IL-6 fusion protein. These studies show the utility of this MAb as a tool for both research and protein purification.

An immunological subtraction approach, surface-epitope masking (SEM), is described that permits the efficient and selective production of monoclonal antibodies (MAbs) reacting with both known and unknown molecules expressed on the cell surface. The tenet underlying SEM involves blocking (masking) of shared antigens between two target populations, a "driver" and a "tester," and using appropriately modified surface-masked "tester" cells to generate MAbs reacting with surface antigens unique to the "tester population" that differentiate the two antigen sources. SEM has been employed to develop MAbs that react with the multidrug resistance surface-expressed P-glycoprotein (MDR-1) and the human interferon-gamma receptor and two potentially novel tumor-associated antigens (TAAs) expressed on the surface of prostate carcinoma and breast carcinoma cells. In principle, the SEM approach provides an uncomplicated and effective means of developing MAbs, which can also be used to identify genes, associated with important cellular processes involved in normal physiology, such as growth, aging, differentiation, and development. In addition, this strategy is amenable to produce MAbs and identify genes associated with specific disease states, including cancer, neurodegeneration, autoimmunity, and infection with pathogenic agents.

Phage display is a molecular technique, whereby genes are displayed in a functional form on the outer surfaces of bacteriophages by fusion to viral coat proteins. The gene product is encoded by a plasmid contained within the virus, which can be recovered and sequenced, linking the genetic information to the function of the protein. Phage display offers a powerful tool for the identification of short peptides or single chain antibodies that can bind and regulate the function of target proteins. One major advantage of phage display lies in its ability to rapidly identify target-specific peptides with pharmacological activity as agonists or antagonists.

Insulin is thought to elicit its effects by crosslinking the two extracellular α-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo , to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.

Establishing cells with an exogenously introduced gene of interest under the inducible control of tetracycline (Tc) initially requires clonal cell lines stably expressing the tetracycline activator (tTA or rtTA). The originally described plasmid vectors expressing tTA/rtTA are driven by the cytomegalovirus (CMV) immediate early (IE) promoter-enhancer, known for its robust activity in a wide spectrum of cell types. While many reports testify to the utility and efficacy of this construct, instances of inexplicable failure to establish cell lines having inducible expression of the cDNA under study are encountered. Spontaneous extinction of CMV promoter activity in cells has been observed in a temporal and cell type-dependent manner. This could be a contributing factor in the failure to establish Tc-responsive cell lines. We here report that a change of the expression cassette to the human elongation factor-1alpha (EF-1alpha) promoter has permitted successful establishment of several inducible cell lines from diverse human tumor tissue origins. We interpret these results to imply that extinction of rtTA (or tTA) expression might be a significant factor in the lack of success in establishing Tc-inducible cell lines. Moreover, the present findings have general relevance to experiments requiring the use of stable cell lines.

Immunoliposomes composed of hydrogenated soy phosphatidylcholine, cholesterol, methoxypoly(ethylene glycol)-distearoyl phosphatidylethanolamine (mPEG-DSPE), and hydrazide-PEG-DSPE (mole ratio, 57:38:3.3:1.7) linked to periodate-oxidized chimerized mouse IgG (C225, anti-human epidermal growth factor receptor) were prepared by an optimized aggregation-free procedure. The antigen-binding activity of the immunoliposomes was well preserved. When injected intravenously into naive rats, the immunoliposomes (approximately 18 IgG per 100 nm liposome) exhibited long circulation times (MRT = 8.5 h, Cl = 0.2 ml/h). Subsequent injections of the immunoliposomes into the same animals resulted in rapid clearance (MRTor = 0.7 h, Clor = 7 ml/h), which was accompanied by a significant increase in anti-C225 specific titers. Upon repeated injection or coinjection with the parent liposomes free C225 consistently exhibited prolonged circulation without any increase in C225-specific antisera, but was cleared quickly when administered into animals that had been pretreated with the immunoliposomes. Screening of the immunoliposome induced antisera against human polyclonal IgG and C225-derived Fab' fragment revealed that the immune response was specifically triggered by the constant human region of C225. These results demonstrate that the preparations of PEG-grafted immunoliposomes are more immunogenic than the free IgG component, which is of profound importance to the antibody-mediated liposomal drug delivery effort.

The selective production of monoclonal antibodies (mAbs) reacting with defined cell surface-expressed molecules is now readily accomplished with an immunological subtraction approach, surface-epitope masking (SEM). Using SEM, prostate carcinoma (Pro 1.5) mAbs have been developed that react with tumor-associated antigens expressed on human prostate cancer cell lines and patient-derived carcinomas. Screening a human LNCaP prostate cancer cDNA expression library with the Pro 1.5 mAb identifies a gene, prostate carcinoma tumor antigen-1 (PCTA-1). PCTA-1 encodes a secreted protein of approximately 35 kDa that shares approximately 40% sequence homology with the N-amino terminal region of members of the S-type galactose-binding lectin (galectin) gene family. Specific galectins are found on the surface of human and marine neoplastic cells and have been implicated in tumorigenesis and metastasis. Primer pairs within the 3' untranslated region of PCTA-1 and reverse transcription-PCR demonstrate selective expression of PCTA-1 by prostate carcinomas versus normal prostate and benign prostatic hypertrophy. These findings document the use of the SEM procedure for generating mAbs reacting with tumor-associated antigens expressed on human prostate cancers. The SEM-derived mAbs have been used for expression cloning the gene encoding this human tumor antigen. The approaches described in this paper, SEM combined with expression cloning, should prove of wide utility for developing immunological reagents specific for and identifying genes relevant to human cancer.

We have constructed two different muteins of interleukin-6 (IL-6) which were expressed in Escherichia coli. Both muteins lack the first 22 N-terminal amino acids of native IL-6 and lack one or the other of the two naturally occurring pairs of cysteines at either position 45 and 51 or position 74 and 84 of IL-6. We found that there was a dramatic increase in the level of IL-6 produced from each mutein clone, compared to the level produced by the wild-type IL-6 clone. We also observed that the yield of soluble and properly refolded mutein IL-6 was highest when the cysteines at position 74 and 84 were left intact. The mutein IL-6 with cysteines at position 74 and 84 was as active as wild-type IL-6 and a lower concentration of the mutein IL-6 was required to reach maximal activity, compared to wild-type IL-6. The mutein IL-6 with cysteines at position 45 and 51 had a much reduced biological activity.

Background Producing monoclonal antibodies against specific targets, including tumor-specific antigens, is a tedious and extremely inefficient process. Purpose Our purpose was to determine whether DNA transfection combined with an immunologic masking tactic could be used to efficiently generate hybridomas that secrete monoclonal antibodies. The quest was for monoclonal antibodies that would react with molecules existing on the surface of genetically altered cells. Methods We developed a masking technique called surface-epitope masking (SEM). The SEM procedure involves the selective blocking of surface antigens present in a genetically engineered cell (referred to as a "tester") with high-titer polyclonal antibodies that have been produced against the untransfected parental cell (referred to as a "driver"). Surface-epitope-masked tester cells were injected into BALB/c mice; immune spleen cells then taken from these mice were fused with myeloma cells. Results This process resulted in the efficient generation of hybridomas that secreted monoclonal antibodies that reacted with cell-surface antigens on transfected tester cells and with additional cell types that expressed the same surface molecules. In one case, CREF-Trans 6 cells were engineered to express a typical multidrug-resistant (MDR) phenotype. Using CREF-Trans 6:MDR cells as a tester cell line, we utilized the SEM procedure to produce monoclonal antibodies that displayed surface reactivity to both CREF-Trans 6:MDR cells and MDR human breast carcinoma (MCF7) cells. In a second case, human prostatic carcinoma CREF-Trans 6 cells, which were DNA transfected and derived from nude mouse tumors, were used as the tester cell line. The SEM procedure was again used to produce monoclonal antibodies. These antibodies were designed to and did react with: (a) tumor-associated antigens on the surface of the original LNCaP cell line used to obtain human prostatic carcinoma DNA, (b) primary and secondary nude mouse transfectants derived from tumors, and (c) two additional human prostatic carcinoma cell lines, DU-145 and PC-3. Conclusions The SEM approach was used for the efficient and selective development of monoclonal antibodies that react with cell-surface molecules with both known and unknown functions. Implications The SEM procedure should be useful in producing monoclonal antibodies and identifying genes associated with important cellular processes, including immunologic recognition, tumorigenesis, metastasis, atypical multidrug resistance, and autoimmune diseases.

This paper describes the use of a novel immune complex (IC) to generate neutralizing monoclonal antibodies to basic fibroblast growth factor. The IC uses a non-neutralizing monoclonal antibody bound to Protein A, itself coated on a solid support, to capture the antigen. Presumably, the capture antibody binds to a region of the antigen distal from the neutralizing site. Animals, immunized with the IC, develop a neutralizing titer and hybridomas producing neutralizing antibodies to basic FGF were obtained. This method can be used to generate neutralizing monoclonals to highly conserved growth factors whenever the antigen can be captured at a site distal to the neutralizing eptiopes.

A clonal cell line (designated MOE CL1) was isolated from the olfactory mucosa of the mouse. A number of growth and biochemical parameters were studied. Our experiments suggest that MOE CL1 cells are olfactory basal cells (undifferentiated neuroblasts).

Olfactory receptor neurons undergo continual replacement with new cells differentiating from a population of basal cells located in the olfactory epithelium. We have previously described the isolation of a cell line from murine olfactory epithelium (MOE CL1) which is now shown to be sensitive to two differentiation promotion agents, dibutyryladenosine 3′, 5′-cyclic monophosphate (db-cAMP) and12-O-tetradecanoylphorbol-13-acetate(TPA). Cells grown in the presence of db-cAMP or TPA, after a lag phase of 72–96 h, became bipolar, with long neurite-like processes. Cells conditioned for at least one passage in db-cAMP or TPA, or cells exposed to a mixture of db-cAMP and TPA, attained a bipolar morphology within 24 h. Cultures grown with db-cAMP, TPA or db-cAMP + TPA showed significant increases in the percentage of process-bearing cells. An increase in population doubling time and decrease in saturation density were also observed in cultures exposed to db-cAMP. Cells exposed to TPA continued to divide at the same rate as control cells, while those treated with db-cAMP + TPA underwent terminal differentiation. Peripheral-type benzodiazepine binding sites, which within the olfactory epithelium are localized on the receptor neurons, were present in homogenate membranes of undifferentiated cells. Cells differentiated by exposure to db-cAMP + TPA for 7 days showed a 65% increase in the density (B max ) of peripheral-type benzodiazepine binding sites. Undifferentiated cells in control cultures showed no spontaneous changes in membrane potential or regenerative responses during current injection. Small, bipolar cells in morphologically differentiated cultures, however, were capable of generating several types of spontaneous changes in membrane potential including slow, regular oscillations; irregular, slow and fast depolarizations; and trains of action potentials.

Interspecific human-mouse and Chinese hamster-mouse hybrids were isolated from polyethylene glycol fused cells by a new half-selection technique employing a structurally modified polyene macrolide antibiotic, amphotericin B methyl ester (AME), and HAT media. Unfused parental cells were killed as a result of innate sensitivity to AME or their genetic deficiency, absence of thymidine kinase (TK-) or hypoxanthine guanine-phosphoribosyl transferase (HGPRT-). In contrast, hybrid colonies were isolated after two to three weeks growth in three or four changes of HAT-AME media and subsequent growth in HAT media alone. The ability of hybrid cells to proliferate using this selective protocol indicates that genetic complementation resulted, and polyene antibiotic resistance was expressed as a dominant phenotypic property in the hybrids. Hybrid selection was dependent on: (1) the number of cells of each parental cell type co-cultivated; (2) the level of polyene antibiotic administered; and (3) the time interval before selection was initiated. The half-selection technique described in this report is simple to use, very effective in eliminating unfused parental cells and increases the potential types of hybrids which can be formed. Only one parental cell type need contain a biochemical defect, whereas the second parental type can be genetically normal.

In the present study we tested the feasibility of utilizing a structurally modified polyene antibiotic, amphotericin B methyl ester (AME), as a half-selection agent for isolating somatic cell hybrids. By using HAT medium supplemented with AME we have isolated interspecific mouse-hamster hybrids from mixed cultures of mouse (TK-C1 ID or HPRT-A9) and hamster (BHK/C 13) cells fused with Sendai virus, lysolecithin or polyethylene glycol. Hybrid cells proliferated and clones were isolated after 2 to 3 weeks growth in three changes of HAT-AME medium and subsequent growth in HAT medium alone. In contrast, genetically deficient parental C1 1D or A9 cells and AME-sensitive BHK/C 13 cells were killed using a similar growth protocol. The described technique is simple, efficient and permits one to use a cell line without a genetic defect in combination with a genetically deficient cell type in hybrid formation.