Requirements for ectopic homologous recombination in mammalian somatic cells.

Ectopic recombination occurs between DNA sequences that are not in equivalent positions on homologous chromosomes and has beneficial as well as potentially deleterious consequences for the eukaryotic genome. In the present study, we have examined ectopic recombination in mammalian somatic (murine hybridoma) cells in which a deletion in the mu gene constant (Cmu) region of the endogenous chromosomal immunoglobulin mu gene is corrected by using as a donor an ectopic wild-type Cmu region. Ectopic recombination restores normal immunoglobulin M production in hybridomas. We show that (i) chromosomal mu gene deletions of 600 bp and 4 kb are corrected less efficiently than a deletion of only 2 bp, (ii) the minimum amount of homology required to mediate ectopic recombination is between 1.9 and 4.3 kb, (iii) the frequency of ectopic recombination does not depend on donor copy number, and (iv) the frequency of ectopic recombination in hybridoma lines in which the donor and recipient Cmu regions are physically connected to each other on the same chromosome can be as much as 4 orders of magnitude higher than it is for the same sequences located on homologous or nonhomologous chromosomes. The results are discussed in terms of a model for ectopic recombination in mammalian somatic cells in which the scanning mechanism that is used to locate a homologous partner operates preferentially in cis.

Role of bone marrow transplantation in 90Y antibody therapy of colon cancer xenografts in nude mice.

The efficacy of bone marrow transplantation (BMT) for the prevention of 90Y toxicity and extension of survival in nude mice with i.p. LS174T carcinomatosis was evaluated. 90Y-labeled monoclonal antibody (MAB) directed against carcinoembryonic antigen (90Y-anti-CEA MAB) at a dose of 120 microCi caused no deaths due to treatment toxicity and increased the duration of animal survival. No long term cures were obtained in these mice. At doses of 160 microCi or more 90Y-anti-CEA MAB led to hematological deaths. Nude mice were given i.p. injections of 10(6) LS174T tumor cells on day 0. On day 7 the mice received 90Y-anti-CEA MAB i.p. at doses of 120-225 microCi. Syngeneic bone marrow cells (10(7) cells) were then injected i.v. into the mice at 1, 3, 5, 7, 10, or 14 days following 90Y treatment. In the absence of BMT, toxic deaths for animals given 175 microCi 90Y were 11 of 24 (46%) with a median survival of 17 days and 13 of 20 (65%) for animals given 225 microCi 90Y with a median survival of 14 days. Animals receiving the same two doses of 90Y and given BMT 5 days following the 90Y treatment showed 0 of 24 (0%) and 0 of 54 (0%) toxicity deaths, respectively. The optimal time of BMT in relation to 90Y therapy was dependent upon the dose of 90Y-anti-CEA MAB (225 microCi, 3-5 days; 175 microCi, 5-14 days). The mean survival in tumor bearing animals was extended from 31.7 +/- 1.2 (SE) to 45.3 +/- 2.0 days by treatment with 120 microCi of 90Y-anti-CEA MAB. By increasing the dose of 90Y-anti-CEA MAB to 225 microCi and undertaking BMT 5 days later the mean survival was further extended to 63.2 +/- 3.6 days (P less than 0.005). BMT administered at the optimal times can prevent toxic deaths and facilitates higher, more effective doses of tumor specific 90Y-MAB.

Mechanisms of tissue uptake and metabolism of radiolabeled antibody--role of antigen: antibody complex formation.

Scintigraphic studies in animals and in humans have demonstrated uptake of radiolabeled antibody by both normal and tumor tissue. Normal tissues most commonly visualized are blood, liver, spleen, kidneys, lymph nodes, bone, and thyroid. A number of factors have been demonstrated to affect the uptake by normal and tumor tissue, including radioisotope properties, immunoglobulin characteristics, antibody specificity, tumor size, vascular permeability, and antigen expression. Clarification of the mechanisms of tumor and normal tissue uptake depends upon comparison of scintigraphic findings with analysis of tissue for such factors as radioactivity, antigen content, and tumor size. One of the major limitations of 111In labeled monoclonal antibody imaging has been extensive 111In uptake by histologically normal liver, especially in a host bearing a large tumor mass. By high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis analysis of liver and blood it can be demonstrated that much of the liver uptake is related to the formation of antigen:antibody complexes. The normal liver intensity can be decreased by inhibition of radiolabeled complex formation. Understanding of the mechanisms of tissue uptake, both normal and tumor, and of radiolabeled antibody metabolism is crucial to the rational planning and use of radioimmunoconjugates for tumor imaging and treatment. Animal and human studies complement one another in examination of these mechanisms.

Mechanism of decreasing liver uptake of 111In-labeled anti-carcinoembryonic antigen monoclonal antibody by specific antibody pretreatment in tumor bearing mice.

The purpose of this study was to examine the mechanism of specific antibody pretreatment for reduction of liver uptake of 111In-labeled monoclonal antibody (MAB). Previous work with an anti-carcinoembryonic antigen (CEA) MAB (T84.66) and LS174T human colon cancer xenografts in nude mice has shown that giving a high dose (0.2 mg) of unlabeled T84.66 in conjunction with the same MAB (T84.66) labeled with 111In (Indacea) significantly lowered the liver uptake of 111In. High performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to assess the radiolabeled components in serum and liver at different times following administration of Indacea in normal and tumor bearing mice. In serum the 111In remained associated with the IgG in both tumor bearing and non-tumor bearing mice. Liver uptake of 111In in mice without tumor was low (8-12% injected dose/g) and both IgG and a low molecular weight metabolite were found in the liver homogenates. Liver uptake in tumor bearing mice increased dramatically (15-40% injected dose/g) with size of tumor and in addition to the IgG and low molecular weight components, a high molecular weight compound was identified. Administration of CEA: Indacea complexes to non-tumor bearing mice produced the same high pressure liquid chromatography and gel patterns as those seen in mice with large (greater than 1 g) tumors. Liver homogenates from tumor bearing mice given specific antibody pretreatment showed the same patterns seen with non-tumor bearing mice (no high molecular weight peak). In conclusion, CEA:Indacea complexes are formed in tumor bearing mice and rapidly cleared by the liver. Specific antibody pretreatment results in the production of unlabeled CEA:MAB complexes causing a reduction in the formation of CEA:Indacea complexes and a lower liver uptake of 111In.

Radioimmunotherapy of human colon carcinomatosis xenograft with 90Y-ZCE025 monoclonal antibody: toxicity and tumor phenotype studies.

Monoclonal antibody ZCE025 recognizes an epitope of the carcinoembryonic molecule (CEA). We have shown that when linked to 90Y, its localization in the tumor was sufficient to result in a significant tumoricidal effect in human colon carcinomatosis grown in the peritoneum of athymic mice. Intraperitoneal tumors were present 7 days after inoculation of the CEA-producing human colon carcinoma cell line LS174T, when the mice received i.p. injections with 40 to 160 microCi of 90Y-labeled ZCE025 or 96.5c (nonspecific monoclonal antibody). The animals that were autopsied 12 days after treatment displayed a significant (P less than 0.001) inhibition of tumor growth when compared to the control animals that received no treatment or similar doses of nonspecific monoclonal antibody. Microscopically, the treated tumors showed extensive radiation effect and they became progressively necrotic until only a rim of viable tissue remained in the periphery of the nodules. CEA expression was practically absent on the newly grown nodules that began to appear 3 weeks after therapy, and remained so 6 weeks thereafter. In contrast, over 80% of the tumor cells from the untreated animals expressed CEA. There was no mortality due to treatment; however, the hematopoietic organs were markedly depleted at the higher doses. The marrow and the spleen recovery began 2 weeks after treatment, and it was completed by the 4th week. No evidence of toxicity was present in any of the other organs examined. These studies suggest that 90Y-ZCE025 therapy results in clonal selection of cells lacking or minimally expressing CEA. The inherent implications of these findings are discussed.

Interferon enhancement of radioimmunotherapy for colon carcinoma.

Recombinant human gamma-interferon (IFN-gamma) has recently been shown to enhance localization of radiolabeled monoclonal antibodies (MAb) to human colon carcinoma xenografts in athymic mice. The present study investigates the ability of gamma-interferon to enhance radioimmunotherapy of a low carcinoembryonic antigen-expressing human colon cancer (WiDr) in athymic mice. Growth curve analysis, antibody localization, and dose estimation studies were performed. A significant tumor growth delay, measured as the time to reach 1.0 g, was noted for animals receiving specific anti-carcinoembryonic antigen 90Y-MAb (ZCE025, 120 microCi) plus IFN-gamma (61.8 days) as compared to animals that received specific 90Y-MAb with phosphate-buffered saline (34.9 days; P less than 0.005). IFN-gamma (100,000 units) was given i.p. every 8 h for 2 days before and 4 days after 90Y-MAb therapy. The time required to reach 1.0 g for animals treated with nonspecific 90Y-MAb (ZME018) was significantly less either with (38.3 days) or without (34.4 days) IFN-gamma. The difference was more apparent when compared to animals receiving IFN-gamma alone (30.0 days) or phosphate-buffered saline alone (28.9 days; P less than 0.001). Increased antibody localization in the tumors of animals treated with IFN-gamma plus specific 90Y-MAb (43.2% injected dose/g) was seen in comparison to animals treated with specific 90Y-MAb without IFN-gamma (18.2% injected dose/g). The estimate of radiation dose delivered to the tumors, based on biodistribution data over time, revealed significantly higher levels in animals treated with specific 90Y-MAb with IFN-gamma (2477 cGy) compared to animals treated without IFN-gamma (1217 cGy). These results provide support for the use of gamma-interferon as an immunomodulating agent prior to radioimmunotherapy.

Testis imaging with 111In-labeled anticarcinoembryonic antigen monoclonal antibody: identification of carcinoembryonic antigen in normal germ cells.

A monoclonal antibody to carcinoembryonic antigen (CEA) labeled with 111In (Indacea) was used to image tumors in patients with colorectal cancer. The anti-CEA antibody has a high affinity (2.6 X 10(10) M-1) for CEA and does not cross-react with normal cross-reacting antigen, biliary glycoprotein-1, or tumor-extracted, CEA-related antigen. During the course of these studies, it was noted that a significant number of male patients (20 of 27, 74%) showed uptake of Indacea in the testes. In order to determine if the Indacea uptake was specific, 20 testicular specimens were analyzed by immunohistological methods using five different anti-CEA CEA monoclonal antibodies recognizing five different epitopes on CEA. In 18 cases (90%) germ cells were uniformly stained by all five antibodies. Fresh frozen testis tissue was homogenized in water and precipitated with 1.0 M perchloric acid. The supernatant contained a CEA-like material as measured by an enzyme immunoassay specific for CEA. The same supernatant was radiolabeled with 125I and immunoprecipitated with anti-CEA monoclonal antibodies. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of the immunoprecipitates revealed a single species (Mr 180,000) which was indistinguishable from CEA. This study documents the first description of CEA in the germ cells of normal testis. The CEA in the testis was accessible to circulating monoclonal antibodies in the majority of male patients tested.

Antigenic sites in carcinoembryonic antigen.

The epitope reactivities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen from 11 different research groups were studied using competitive solid-phase immunoassays. About 60% of all possible combinations of Mabs as inhibitors and as the primary binding antibody were investigated. The inhibition data were analyzed by a specially developed computer program "EPITOPES" which measures concordance and discordance in inhibition patterns between Mabs. The analysis showed that 43 of the 52 Mabs (83%) could be classified into one of five essentially noninteracting epitope groups (GOLD 1-5) containing between four and 15 Mabs each. The epitopes recognized by the Mabs belonging to groups 1 to 5 were peptide in nature. With one or two possible exceptions non-classifiable Mabs were either directed against carbohydrate epitopes (4 Mabs) or were inactive in the tests used. Within epitope groups GOLD 1, 4, and 5 two partially overlapping subgroups were distinguished. Mabs with a high degree of carcinoembryonic antigen specificity generally belonged to epitope groups GOLD 1 and 3.

Estimation of monoclonal antibody-associated 90Y activity needed to achieve certain tumor radiation doses in colorectal cancer patients.

In these measurements, we quantitated, via surgical samples, human primary tumor uptake of the anti-carcinoembryonic antigen monoclonal antibody T84.66. Uptake was measured in units of percentage of injected dose/kg with 111In as the radiolabel. All 11 colorectal lesions were nonnecrotic and were visualized upon scanning. Tumor volume was calculated using the three orthogonal dimensions as described by pathology. Uptake mean +/- SD was 6.55 +/- 3.55% injected dose/kg with a range of 1.2 to 10.4% injected dose/kg. Lesion mean volume was 36 cm3 with a range of 1.5 to 304 cm3. Using mean values, assuming no biological clearance and that the biodistribution of the monoclonal is independent of its radiolabel, the predicted human tumor 90Y beta dose was 0.12 Gy/mCi. Therefore a 10-Gy tumor dose would require 83 mCi of i.v. activity. Using least and highest uptake results, requisite activity values were 3-fold larger and smaller respectively. Thus, there was approximately an order of magnitude variation in the amount of 90Y predicted to achieve a given tumor dose in colorectal cancer patients. Murine and human uptake values were consistent if lesion mass and carcinoembryonic antigen content were taken into account.

Presurgical imaging with indium-labeled anti-carcinoembryonic antigen for colon cancer staging.

Over a 4-year period, 108 patients with known or suspected colorectal cancer were studied by radioimmunoconjugate scintigraphy prior to operative procedures. Study subjects received 0.2 to 40 mg i.v. of murine anti-carcinoembryonic antigen monoclonal antibody labeled with 2-5 mCi of 111In (Indacea). Resected tissues were analyzed for 111In and carcinoembryonic antigen content. Tumor, liver, and draining lymph nodes had over 10% injected dose/kg compared to less than 2.5% injected dose/kg for other normal tissues. Primary tumors that were successfully imaged were significantly larger and had higher 111In and carcinoembryonic antigen content. In 54 patients, primary tumors were visualized with a sensitivity of 78%. Hepatic metastases (58 patients) were visualized as negative filling defects (sensitivity, 45%). Extrahepatic (intraabdominal) metastases (25 patients) were visualized (sensitivity, 48%) as areas of increased uptake. Extraabdominal metastases were uncommon (10 patients; sensitivity, 80%). Of 56 patients with known or suspected hepatic metastases who presented with no evidence of extrahepatic disease by conventional tests (X-ray, computerized tomographic scan), 20 (36%) were documented to have extrahepatic metastases at exploratory surgery and 10 of these (50%) had the extrahepatic disease localized by the Indacea scan. The management of these 10 patients was, or could have been, modified by the scan findings and unnecessary surgery eliminated.

Effect of specific antibody pretreatment on liver uptake of 111In-labeled anticarcinoembryonic antigen monoclonal antibody in nude mice bearing human colon cancer xenografts.

Administration of a large dose (0.2 mg) of unlabeled specific anticarcinoembryonic antigen (anti-CEA) monoclonal antibody (MAB) to nude mice bearing LS174T human colon cancer xenografts significantly decreased normal liver uptake of 111In-labeled anti-CEA MAB (Indacea). Mice bearing tumors of approximately 1 g showed liver accumulation of indium-111 at 48 h following injection of 2 micrograms/10 microCi Indacea of 33.8 +/- 1.5% injected dose per gram (%ID/g) (N = 25). Treatment with 0.2 mg unlabeled anti-CEA MAB reduced this to 8.9 +/- 0.5% ID/g (N = 22; P less than 0.001). The dose of pretreatment was found to be critical. Increasing the amount of unlabeled MAB to 2.0 mg did not significantly improve the liver level of indium-111, but did compromise the tumor uptake of Indacea (15.9 +/- 1.3 versus 12.4 +/- 0.4% ID/g; P less than 0.05). Lowering the dose of pretreatment 10-fold resulted in increased (P less than 0.001) liver uptake of the label (26.5 +/- 2.8% ID/g). The unlabeled anti-CEA MAB treatment given as a single dose or fractionated over several days gave the same results. The decrease in liver uptake was the same for i.v. administration of the unlabeled MAB given 1 week prior to Indacea injection or mixed together with Indacea. With i.p. administration, simultaneous injection of the unlabeled MAB with Indacea was not as effective as pretreatment (20 min to 7 days) in decreasing the liver uptake of 111In (P less than 0.05). Epitope specificity and affinity were shown to be important considerations in the choice of MAB combinations used for pretreatment and imaging. Pretreatment with nonspecific MAB was ineffective in decreasing liver uptake of Indacea.

High-specific-activity 111In-labeled anticarcinoembryonic antigen monoclonal antibody: improved method for the synthesis of diethylenetriaminepentaacetic acid conjugates.

A new method has been developed for conjugating diethylenetriaminepentaacetic acid (DTPA) to proteins using the N-hydroxysuccinimide active ester of DTPA. The DTPA-active ester was prepared using diisopropylcarbodiimide in a simple single step synthesis. DTPA-conjugated proteins were prepared by adding the DTPA-active ester reaction mixture to protein solutions (5 mg/ml) buffered at pH 7.0 and purified by Sephadex G-50 chromatography. A monoclonal antibody directed against carcinoembryonic antigen was reacted with four different amounts of the DTPA-active ester. Solid-phase enzyme immunoassay showed that the immunological activity of the antibody conjugate was not altered when the active ester: antibody molar ratio was 36:1 or 72:1; however, it decreased when the ratio was 180:1 or 360:1. The antibody heavy and light chains had slightly decreased electrophoretic mobilities when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a result consistent with the covalent attachment of DTPA to the protein. Sephadex G-200 chromatography showed that the native and conjugated antibodies were the same size. When the DTPA-conjugated antibody was incubated with 10, 50, and 100 microCi of 111In/micrograms of protein, specific activities of 9.8, 43.1, and 56.3 microCi/micrograms were obtained. Enzyme immunoassay and radioimmunoassay of the 111In-labeled antibody showed that it retained its full immunological activity. The high specific activity of the 111In-labeled antibody makes it suitable for imaging carcinoembryonic antigen-bearing tumors using low doses of antibody.

High-specific-activity 111In-labeled anticarcinoembryonic antigen monoclonal antibody: biodistribution and imaging in nude mice bearing human colon cancer xenografts.

Tumor imaging and biodistribution of an indium-labeled monoclonal antibody (MAB) to carcinoembryonic antigen (CEA) [anti-CEA MAB-diethylenetriaminepentaacetic acid (DTPA)-111In] have been investigated using LS174T human colon cancer xenografts in nude mice. Antibody specificity, dose, and specific activity were examined with respect to tumor uptake and quality of scintiscans at different times following injection. The CEA-bearing LS174T tumors were imaged specifically with anti-CEA MAB-DTPA-111In. Using 62.5 ng of indium-labeled MAB (50 microCi/micrograms) the ratio of activity in tissue expressed as a percentage of the total radioactive dose injected into the animal per gram tissue for tumor:blood increased from 0.66 +/- 0.02 (SE) at 1 h to 14.8 +/- 1.1 at 72 h. Scintiscan quality improved with the rise in tumor:blood ratio until 48 h. At longer intervals insufficient counts remained for imaging. The tumor:blood ratio and the scintiscan quality were not improved by increasing the MAB dose to 625 or 6250 ng but good images were obtained at longer times postinjection. By decreasing the 111In from 50 to 10 microCi/micrograms of MAB, the unbound 111In was decreased from 7 microCi/micrograms (14%) to 0.2 microCi/micrograms (2%). Even with the lower specific activity (9.8 microCi/micrograms) of the 10-microCi/micrograms preparation, scintiscan quality at the 62.5-ng dose was maintained. This anti-CEA MAB-DTPA-111In preparation was stable, retained immunological activity, did not require column chromatography to remove unbound 111In, was specific for a CEA-bearing tumor, and was effective for tumor imaging over a wide range of antibody doses (3 to 300 micrograms MAB/kg body weight). This anti-CEA MAB-DTPA-111In preparation is feasible and practical for imaging CEA-bearing tumors in humans.

Preoperative imaging of colorectal carcinoma with 111In-labeled anticarcinoembryonic antigen monoclonal antibody.

Patients with primary, recurrent, or metastatic colorectal adenocarcinoma were given injections of 200 micrograms of anticarcinoembryonic antigen (CEA) monoclonal antibody labeled with 2 mCi of 111In (Indacea). Patients were imaged at 24 and 48 h. Celiotomy was performed on 40 patients between 3 and 17 days post-Indacea injection. Of 16 primary tumors, 11 (69%) were imaged. Of six extrahepatic recurrences, none was imaged. Intrahepatic metastases were visualized as negative images in 10 of 24 (42%) patients. On the basis of the activity in tissue expressed as a percentage of the total radioactive dose per kg injected into the patient (% ID/kg), extrahepatic tumors that were imaged using Indacea had a significant uptake of radiolabel in the tumor [5.99 +/- 0.91% ID/kg (SE)] and in the associated normal mesenteric lymph nodes (12.0 +/- 2.4% ID/kg). The CEA content of these tumors was high (13.3 +/- 4.7 micrograms/g), and, histologically, the CEA was located primarily apically or intraluminally. Intrahepatic tumor imaging correlated only with tumor size. The greatest Indacea uptake was seen in normal liver (22.1 +/- 3.2% ID/kg). Low Indacea uptake was seen in fat (0.21 +/- 0.05% ID/kg) and bowel wall (1.11 +/- 0.17% ID/kg). In conclusion, Indacea imaging of colorectal carcinoma is specific for high concentrations of accessible CEA in CEA-bearing tumors or in lymph nodes draining these tumors. The successful clinical use of monoclonal antibodies for tumor imaging and therapy will require careful selection of patients for a number of antigen-related parameters including antigen content and distribution in tumors. This information will only come from careful correlation between image results and tissue analysis. High uptake by normal liver tissue is the major unresolved problem with labeled antibody imaging.

Primary structure, expression and chromosomal locus of a human homolog of rat ERK3.

We report the cloning and characterization of a human cDNA encoding a novel homolog of rat extracellular signal-regulated kinase 3 (ERK3). The cDNA encodes a predicted protein of 721 amino acids which shares 92% amino acid identity with rat ERK3 over their shared length. Interestingly, the human protein contains a unique extension of 178 amino acids at its carboxy terminal extremity. The human ERK3 protein also displays various degrees of homology to other members of the MAP kinases family, but does not contain the typical TXY regulatory motif between subdomains VII and VIII. Northern blot analysis revealed that ERK3 mRNA is widely distributed in human tissues, with the highest expression detected in skeletal muscle. The human ERK3 gene was mapped by fluorescence in situ hybridization to chromosome 15q21, a region associated with chromosomal abnormalities in acute nonlymphoblastic leukemias. This information should prove valuable in designing studies to define the cellular function of the ERK3 protein kinase.

Isolation and characterization of the human UGT2B15 gene, localized within a cluster of UGT2B genes and pseudogenes on chromosome 4.

Glucuronidation is a major pathway of androgen metabolism and is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. UGT2B15 and UGT2B17 are 95% identical in primary structure, and are expressed in steroid target tissues where they conjugate C19 steroids. Despite the similarities, their regulation of expression are different; however, the promoter region and genomic structure of only the UGT2B17 gene have been characterizedX to date. To isolate the UGT2B15 gene and other novel steroid-conjugating UGT2B genes, eight P-1-derived artificial chromosomes (PAC) clones varying in length from 30 kb to 165 kb were isolated. The entire UGT2B15 gene was isolated and characterized from the PAC clone 21598 of 165 kb. The UGT2B15 and UGT2B17 genes are highly conserved, are both composed of six exons spanning approximately 25 kb, have identical exon sizes and have identical exon-intron boundaries. The homology between the two genes extend into the 5'-flanking region, and contain several conserved putative cis-acting elements including Pbx-1, C/EBP, AP-1, Oct-1 and NF/kappaB. However, transfection studies revealed differences in basal promoter activity between the two genes, which correspond to regions containing non-conserved potential elements. The high degree of homology in the 5'-flanking region between the two genes is lost upstream of -1662 in UGT2B15, and suggests a site of genetic recombination involved in duplication of UGT2B genes. Fluorescence in situ hybridization mapped the UGT2B15 gene to chromosome 4q13.3-21.1. The other PAC clones isolated contain exons from the UGT2B4, UGT2B11 and UGT2B17 genes. Five novel exons, which are highly homologous to the exon 1 of known UGT2B genes, were also identified; however, these exons contain premature stop codons and represent the first recognized pseudogenes of the UGT2B family. The localization of highly homologous UGT2B genes and pseudogenes as a cluster on chromosome 4q13 reveals the complex nature of this gene locus, and other novel homologous UGT2B genes encoding steroid conjugating enzymes are likely to be found in this region of the genome.

Intrachromosomal recombination between well-separated, homologous sequences in mammalian cells.

In the present study, we investigated intrachromosomal homologous recombination in a murine hybridoma in which the recipient for recombination, the haploid, endogenous chromosomal immunoglobulin mu-gene bearing a mutation in the constant (Cmu) region, was separated from the integrated single copy wild-type donor Cmu region by approximately 1 Mb along the hybridoma chromosome. Homologous recombination between the donor and recipient Cmu region occurred with high frequency, correcting the mutant chromosomal mu-gene in the hybridoma. This enabled recombinant hybridomas to synthesize normal IgM and to be detected as plaque-forming cells (PFC). Characterization of the recombinants revealed that they could be placed into three distinct classes. The generation of the class I recombinants was consistent with a simple unequal sister chromatid exchange (USCE) between the donor and recipient Cmu region, as they contained the three Cmu-bearing fragments expected from this recombination, the original donor Cmu region along with both products of the single reciprocal crossover. However, a simple mechanism of homologous recombination was not sufficient in explaining the more complex Cmu region structures characterizing the class II and class III recombinants. To explain these recombinants, a model is proposed in which unequal pairing between the donor and recipient Cmu regions located on sister chromatids resulted in two crossover events. One crossover resulted in the deletion of sequences from one chromatid forming a DNA circle, which then integrated into the sister chromatid by a second reciprocal crossover.

Effects of recombinant human gamma-interferon on carcinoembryonic antigen expression of human colon cancer cells.

The effects of human recombinant gamma-interferon (gamma-IFN) on the levels of carcinoembryonic antigen (CEA) expression were investigated in vitro in three human colon adenocarcinoma cell lines (WiDr, HT29, and SW403). Subconfluent cultures were exposed continuously to IFN at concentrations of 1-1,000 antiviral units/ml for up to 6 consecutive days. IFN resulted in a significant increase in CEA levels when assayed by cellular enzyme-linked immunosorbent assay (ELISA), with higher concentrations and longer exposure times resulting in greater CEA enhancement. A three to five-fold enhancement of CEA was observed after 5-6 days of continuous exposures at concentrations of 100-1,000 antiviral units/ml. CEA levels returned to baseline over a 4-day period after discontinuation of IFN. Levels of IFN that resulted in CEA enhancement also resulted in cell growth inhibition, with a direct correlation observed. Flow cytometric studies, which evaluated changes in CEA membrane expression of only the viable cells remaining after IFN exposure, gave similar results to cellular ELISA. Quantitative CEA ELISA, which quantitated changes in total cellular CEA content, demonstrated greater increase in CEA than predicted by cellular ELISA. Continuous IFN exposures for 5-6 days at 1,000 U/ml led to a 96-, 26-, and 5-fold increase in total CEA for the WiDr, HT29, and SW403 cell lines, respectively. WiDr cells exposed to daily 6-h IFN pulses demonstrated intermediate increases in CEA compared with cells exposed continuously to IFN.(ABSTRACT TRUNCATED AT 250 WORDS)

Gamma-interferon enhancement of carcinoembryonic antigen expression in human colon carcinoma xenografts.

Athymic nu/nu mice bearing a subcutaneous human colon cancer xenograft (WiDr, low CEA expression) were treated with gamma-interferon (gamma IFN) at varying doses, frequencies, and periods of duration. CEA content (micrograms/g) and uptake of radiolabeled anti-CEA monoclonal antibody (MAB) (percent injected dose per gram, % ID/g) were measured at 48 h following administration of the MAB, The optimal enhancement of tumor CEA content and tumor localization of [111In] anti-CEA monoclonal antibody (MAB) was seen at gamma IFN doses of 100,000 U i.p. every 8 h for 4 days (4.7 micrograms/g; 29% ID/g) compared to control animals (0.9 micrograms/g; 10% ID/g). The effects of gamma IFN on CEA content and MAB localization were less pronounced when administered (a) at lower doses: 5,000 to 50,000 U i.p. every 8 h, (b) at varying frequencies: 300,000 U/day delivered in divided doses every 4 or 24 h, or (c) for varying periods: 2 or 6 days of therapy. In each case, the biologic effects on tumor CEA content and uptake of [111In]MAB correlated closely with the serum gamma IFN level. Therefore, we conclude that enhancement of in vivo CEA expression by gamma IFN may have clinical relevance for tumor imaging and therapy using radiolabeled monoclonal antibodies.

Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay.

Enzyme-linked immunoadsorbent assay (EIA) has widespread use for the measurement of antibody concentration. The affinity constant (Kaff) of the antibody has an effect upon the quantification by EIA. It is thus important to be able to measure Kaff by solid-phase EIA. Based upon the Law of Mass Action and using serial dilutions of both antigens (coating the plate) and antibody, Kaff has been measured by EIA. A microtiter plate was coated with antigen (Ag) and then incubated with monoclonal antibody (Ab). The plate was sequentially incubated with a second enzyme-antibody conjugate (EAC) and with the enzyme substrate. The amount of Ab adherent to Ag on the plate [Ag Ab] and [Ag2 Ab] was reflected by the enzyme product measured by OD. The use of serial dilutions of Ab resulted in a sigmoid curve of OD versus logarithm of total Ab added to the well. Comparison of the OD at the upper plateau (OD-100) for different antibodies was a reflection of the relative number of epitopes on the Ag that were identified by the different antibodies, provided excessive EAC was used. [Ab]t and [Ab']t were the measurable total antibody concentrations in the wells at OD-50 and OD-50' for plates coated with [Ag] and [Ag'], respectively. [Ag] and [Ag'] were not true antigen concentrations, but were a measure of antigen density on the plate. For [Ag'] = [Ag]/2, Kaff = 1/2(2[Ab']t-[Ab]t. Using five different anti-CEA antibodies and different proportions of CEA in the coating solution, Kaff was measured. Kaff determined by EIA correlated well with Kaff measured by soluble phase inhibition assay. This EIA method of estimation of Kaff is simple, rapid, and reliable.