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C L Chen

Publications and source records attributed to C L Chen.

At least 145 records · Page 8Linked to original sources
PubMed1999

The fas and fas ligand pathways in liver allograft tolerance.

The Fas and Fas ligand (Fas/FasL) pathways may play a central role in cytotoxicity or immunoregulation in liver transplantation. Here, in an attempt to examine the role of Fas/FasL on drug-free tolerance, we measured mRNA levels of Fas/FasL in livers by reverse transcriptase-polymerase chain reaction (RT-PCR), and also protein levels of Fas/FasL in livers by immunohistochemistry and in serum by dot blot assay. PVG recipients bearing DA livers showed serious rejection between post-operative (POD) days 7 and 14, but this rejection was naturally overcome without any immunosuppression. Fas gene and protein products were expressed on almost every cell in livers taken from naive rats, and at any time point in both syngeneic and allogeneic orthotopic liver transplantation (OLT) rats. In contrast, FasL mRNA in DA livers was detectable at POD 2, peaked at POD 14, and declined at POD 63 in allogeneic OLT (DA-PVG). Although the FasL gene was detectable in isografts at POD 14, its expression was much lower than in allografts. The time course and localization of FasL expression indicated that the expression of FasL gradually switched from infiltrating cells to hepatocytes when the rejection was naturally overcome and tolerance was induced in this OLT model. Soluble Fas could constitutively be detected at any time point in the serum of the tolerogenic OLT (DA-PVG) rats and was not diminished during the rejection phase. Soluble FasL peaked at POD 14 in allogeneic OLT, while sFasL was significantly lower in the serum of normal and syngeneic OLT rats. These findings suggest that the Fas and FasL pathways, including soluble forms, may contribute to the control of the immune response in this drug-free tolerance OLT model.

Animals↗
PubMed1999

Immunization with a synthetic peptide conjugate derived from the N-terminal sequence of either the beta-chain of haemoglobin or the immunosuppressive protein (reOLT 4) reduces the litter size of pregnant rats.

A synthetic peptide conjugate based on the N-terminal sequence of a 10 000 MW immunosuppressive serum protein (reOLT 4) was used to immunize female Lewis rats prior to mating, in order to determine whether blocking this protein had an effect on pregnancy. The N-terminal sequence of (reOLT 4) has close sequence homology to the beta-chain of rat haemoglobin so a peptide conjugate based on the N-terminal sequence of this protein was also used to immunize female Lewis rats. Controls included animals that were not immunized and animals that received the peptide carrier, diphtheria toxoid (DT). No statistical differences were found in gestation time or litter sizes in these groups. Differences were, however, evident between these groups and animals that received DT-(reOLT 4) (group 4) or the DT-beta-chain haemoglobin (group 5). There were no statistical differences in litter size or gestation time for group 4 when compared with group 5. Enzyme-linked immunosorbent assay and dot-blot analysis revealed that rats from both groups also had strong responses against DT, the peptide conjugate they were immunized with and the corresponding full-length protein. In both cases, animals from group 4 and group 5 had weak responses to the peptide that they did not receive, together with lower erythrocyte counts and haematocrits, and elevated heart to body weight ratios. Additionally, antibody purified on a (reOLT 4) immunoaffinity column was capable of binding to rat erythrocytes. A second investigation comparing anaemia prior to fertilization and maintained anaemia over the gestation period revealed that only the latter was capable of decreasing litter size to the same degree as obtained for groups 4 and 5. We conclude that for groups 4 and 5 it is the autoimmune effect of continual anaemia over the gestation period, mediated by autoantibodies, which results in the observed lower litter size.

Animals↗
PubMed1999

Safrole-like DNA adducts in oral tissue from oral cancer patients with a betel quid chewing history.

Betel quid (BQ) chewing has been associated with an increased risk of oral squamous cell carcinoma (OSCC) and oral submucous fibrosis (OSF). Piper betel inflorescence, which contains 15 mg/g safrole, is a unique ingredient of BQ in Taiwan. Chewing such prepared BQ may contribute to safrole exposure in human beings (420 microM safrole in saliva). Safrole is a known rodent hepatocarcinogen, yet its carcinogenicity in human beings is largely undetermined. In this study, using a (32)P-post-labeling method, we have found a high frequency of safrole-like DNA adducts in BQ-associated OSCC (77%, 23/30) and non-cancerous matched tissue (NCMT) (97%, 29/30). This was in contrast to the absence (< 1/10(9) nucleotides) of such adducts in all of non-BQ-associated OSCC and their paired NCMT (P < 0.001). Six of seven OSF also exhibited the same safrole-like DNA adduct. The DNA adduct levels in OSF and NCMT were significantly higher than in OSCC (P < 0.05). Using co-chromatography and rechromatography techniques, we further demonstrated that these adducts were identical to synthetic safrole-dGMP adducts as well as DNA adducts from 1'-hydroxysafrole-treated HepG2 cells. These results suggest that safrole forms stable safrole-DNA adducts in human oral tissue following BQ chewing, which may contribute to oral carcinogenesis.

Areca↗
PubMed1999

Synthesis, characterization and preclinical formulation of a dual-action phenyl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07) with potent anti-HIV and spermicidal activities.

In a systematic effort to develop a microbicide contraceptive capable of preventing transmission of human immunodeficiency virus (HIV), as well as providing fertility control, we have previously identified novel phenyl phosphate derivatives of zidovudine (ZDV) with 5-halo 6-alkoxy substitutions in the thymine ring and halo substitution in the phenyl moiety respectively. Here, we describe the synthesis, characterization, and successful preclinical formulation of our lead compound, 5-bromo-6-methoxy-3'-azidothymidine-5'-(p-bromophenyl) methoxyalaninyl phosphate (WHI-07), which exhibits potent anti-HIV and sperm immobilizing activities. The anti-HIV activity of WHI-07 was tested by measuring viral p24 antigen production and reverse transcriptase activity as markers of viral replication in HIV-1 infected human peripheral blood mononuclear cells (PBMC). WHI-07 inhibited replication of HIV in a concentration-dependent fashion with nanomolar IC50 values. The effects of WHI-07 on human sperm motion kinematics were analysed by computer-assisted sperm analysis (CASA), and its effects on sperm membrane integrity were examined by confocal laser scanning microscopy (CLSM), and high-resolution low-voltage scanning electron microscopy (HR-LVSEM). WHI-07 caused cessation of sperm motility in a concentration- and time-dependent fashion. The in-vitro cytotoxicities of WHI-07 and nonoxynol-9 (N-9) were compared using normal human ectocervical and endocervical epithelial cells by the MTT cell viability assay. Unlike N-9, WHI-07 had no effect upon sperm plasma and acrosomal membrane integrity. N-9 was cytotoxic to normal human ectocervical and endocervical cells at spermicidal doses, whereas WHI-07 was selectively spermicidal. The in-vivo vaginal absorption and vaginal toxicity of 2% gel-microemulsion of WHI-07 was studied in the rabbit model. The sperm immobilizing activity of WHI-07 was 18-fold more potent than that of N-9. Over a 10 day period, there was no irritation or local toxicity to the vaginal epithelia or systemic absorption of WHI-07. Therefore, as a potent anti-HIV agent with spermicidal activity, and lack of mucosal toxicity, WHI-07 may have the clinical potential to become the active ingredient of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.

Acrosome Reaction↗
PubMed1999

Functional and transcriptional analyses of a fengycin synthetase gene, fenC, from Bacillus subtilis.

A 37-kb DNA fragment containing five fengycin synthetase genes, including fenC, fenD, fenE, fenA, and fenB, was cloned and sequenced. Among these genes, fenC encodes a fengycin synthetase 2,560 amino acids long with an estimated molecular mass of 287 kDa. This protein contains two amino acid activation modules, FenC1 and FenC2, which activate L-glutamic acid and L-ornithine, respectively. Primer extension, using mRNA isolated from the log-phase cells, identified a transcription start site located 86 nucleotides upstream from the initiation codon of fenC, implying that a promoter is located upstream from the start site. Primer extension using total RNA isolated from stationary-phase cells also identified a transcription start site located 61 nucleotides upstream from the initiation codon of fenC. Gene fusion studies demonstrated that in nHA medium, the cells transcribe the fengycin synthetase genes at two different stages of cell growth. The promoter is active during the log phase, and the activity reaches the highest level during the late log phase. The activity decreases sharply but is maintained at a low level for approximately 24 h after cells enter the early stationary phase. The results of this investigation also suggest that the transcription of fenC is positively regulated during the late log phase. Results presented herein provide further insight into fengycin synthesis by B. subtilis F29-3.

Adenine↗
PubMed1999

Clinical pharmacokinetics of the CD19 receptor-directed tyrosine kinase inhibitor B43-Genistein in patients with B-lineage lymphoid malignancies.

The authors examined the pharmacokinetics of the CD19 receptor-directed tyrosine kinase inhibitor B43-Genistein in 17 patients (4 children, 13 adults) with B-lineage lymphoid malignancies, including 12 patients with acute lymphoblastic leukemia (ALL) and 5 patients with non-Hodgkin's lymphoma (NHL). The immunoconjugate was administered intravenously as a 1-hour continuous infusion at a dose level of either 0.1 mg/kg (N = 12) or 0.18 mg/kg (N = 5), and the plasma concentration-time data were modeled by using the WinNonlin program to estimate the pharmacokinetic parameters. Pharmacokinetic analyses revealed a plasma half-life of 19 +/- 4 hours, mean residence time of 22 +/- 4 hours, and a systemic clearance of 18 +/- 2 mL/h/kg. The average (mean +/- SEM) values for the maximum plasma concentration Cmax, volume of distribution at steady state (Vss), and area under curve (AUC) were 1092 +/- 225 ng/ml, 291 +/- 37 mL/kg, and 9987 +/- 2021 micrograms x h/L, respectively. The AUC values were higher at the 0.18 mg/kg dose level than at the 0.1 mg/kg dose level (16,848 +/- 5118 micrograms x h/L vs. 7128 +/- 1156 micrograms x h/L, p = 0.009). Patients with ALL had a significantly larger volume of distribution at steady state (332 +/- 47 mL/kg vs. 191 +/- 12 mL/kg, p = 0.04), faster clearance (21 +/- 3 mL/h/kg vs. 11 +/- 2 mL/h/kg, p = 0.03), and lower dose-corrected AUC than patients with NHL (6010 +/- 836 micrograms x h/L vs. 12,044 +/- 2707 micrograms x h/L, p = 0.006). There was a trend toward faster clearance rates (23 +/- 4 mL/h/kg vs. 16 +/- 3 mL/h/kg, p = 0.1), shorter elimination half-lives (5.7 +/- 3.6 hours vs. 13 +/- 8.8 hours, p = 0.1), and shorter mean residence times (11 +/- 3 hours vs. 25 +/- 5 hours, p = 0.08) for non-Caucasian patients as compared to Caucasian patients. When compared to adult patients, pediatric patients showed a significantly larger volume of distribution at steady state (418 +/- 82 mL/kg vs. 252 +/- 34 mL/kg, p = 0.02) and a longer elimination half-lives (18.4 +/- 13.6 hours vs. 8.7 +/- 6.7 hours, p = 0.04). The pharmacokinetics of B43-Genistein was not affected by the gender of the patients or by bone marrow transplantation in past medical history. Overall, B43-Genistein showed favorable pharmacokinetics in this heavily pretreated leukemia/lymphoma patient population, which is reminiscent of its recently reported favorable pharmacokinetics in cynomolgus monkeys. To our knowledge, this is the first clinical pharmacokinetics study of a tyrosine kinase inhibitor containing immunoconjugate.

Adolescent↗
PubMed1999

Synthesis of dual function (5R,6R)- and (5S,6S)-5-bromo-6-methoxy-5,6-dihydro-AZT-5'-(para-bromophenyl methoxyalaninyl phosphate) as novel spermicidal and anti-HIV agents.

We synthesized a novel compound, 5-bromo-6-methoxy-5,6-dihydro-AZT-5'- (p-bromophenyl methoxyalaninyl phosphate), which had an EC50 value of 5 microM in sperm motility assays. This is > 1 log10 better than that of the detergent spermicide nonoxynol-9 (EC50 81 microM). The compound also displayed a potent anti-human immunodeficiency virus (HIV) activity with an IC50 value of 0.005 microM in HIV replication assays, which was virtually identical to that of AZT (IC50 0.006 microM) and > 2 log10 more potent than that of nonoxynol-9 (IC50 2.2 microM). The promising results reported herein recommend the further development of the dual function 5-halo-6-alkoxyl-5,6-dihydro-AZT derivatives as a new class of vaginal contraceptives capable of preventing the sexual transmission of HIV while providing fertility control for women who are at high risk of acquiring HIV by heterosexual transmission. These dual function 5-halo-6-alkoxyl-5,6-dihydro-AZT derivatives may also have utility in curbing domestic and wildlife animal retroviral transmissions.

Anti-HIV Agents↗
PubMed1999

Transhepatic artery chemoembolization for liver metastases of primary retroperitoneal endodermal sinus tumor. A case report.

BACKGROUND: Primary retroperitoneal endodermal sinus tumors (EST) are extremely rare and highly malignant. Hepatic metastases from EST are often very difficult to manage and carry a dismal prognosis. Transhepatic artery chemoembolization has been widely applied in primary unresectable hepatoma and had been reported to have antitumor activity for some metastatic tumors. We report a case in which transhepatic artery chemo-embolization was employed to control hepatic metastases from EST. CASE: A 35-year-old woman presented to our service with primary retroperitoneal EST associated with multiple hepatic metastases. The patient underwent aggressive debulking surgery followed by a combination chemotherapy regimen of cisplatin, vinblastine and bleomycin. Transhepatic artery chemoembolization with adriamycin, lipoidol and gelfoam was subsequently employed to treat hepatic metastases. CONCLUSION: The combination of transhepatic artery chemoembolization and systemic chemotherapy after surgical resection of primary retroperitoneal EST was effective in controlling the hepatic disease in this case.

Adult↗
PubMed1999

Expression of nm23 in the primary tumor and the metastatic regional lymph nodes of patients with gastric cardiac cancer.

Tumor recurrence and distant metastasis are major causes of treatment failure in gastric cardiac cancer (GCC). Rapid growth of tumor cells and reduced expression of nm23, a metastatic suppressor gene, in tumor cells have been suggested as two important mechanisms for disease progression of GCC. Therefore, to determine the prognostic value of nm23 expression in GCC, we used immunohistochemistry to examine the expression of nm23 in the pathological sections of both gastric cancer and metastatic lymph nodes from 24 stage III patients. Twenty-two patients had total gastrectomy, and two patients had proximal subtotal gastrectomy with a D2 dissection. Postoperative adjuvant therapy was provided, and the clinical responses were followed routinely. Clinical correlation was evaluated by chi2 with Fisher's exact test and survival by log-rank test. Our results show that the reduced nm23 expression in the primary tumor and in the nodal metastasis is the most useful marker for the poor prognosis of GCC following surgery.

Adult↗
PubMed1999

In vivo toxicity and pharmacokinetic features of the janus kinase 3 inhibitor WHI-P131 [4-(4'hydroxyphenyl)-amino-6,7- dimethoxyquinazoline.

4-(4'Hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131) is a potent and selective inhibitor of the Janus kinase 3, which triggers apoptosis in human acute lymphoblastic leukemia (ALL) cells. In this preclinical study, we evaluated the pharmacokinetics and toxicity of WHI-P131 in rats, mice, and cynomolgus monkeys. Following i.v. administration, the terminal elimination half-life of WHI-P131 was 73.2 min in rats, 103.4 min in mice, and 45.0 min in monkeys. The i.v. administered WHI-P131 showed a very wide tissue distribution in mice. Following i.p. administration, WHI-P131 was rapidly absorbed in both rats and mice, and the time to reach the maximum plasma concentration (tmax) was 24.8 min in rats and 10.0 min in mice. Subsequently, WHI-P131 was eliminated with a terminal elimination half-life of 51.8 min in rats and 123.6 min in mice. The estimated i.p. bioavailability was 95% for rats, as well as for mice. WHI-P131 was quickly absorbed after oral administration in mice with a tmax of 5.8 min, but its oral bioavailability was relatively low (29.6%). The elimination half-life of WHI-P131 after oral administration was 297.6 min. WHI-P131 was not acutely toxic to mice at single i.p. bolus doses ranging from 0.5-250 mg/kg. Two cynomolgus monkeys treated with 20 mg/kg WHI-P131 and one cynomolgus monkey treated with 100 mg/kg WHI-P131 experienced no side effects. Plasma samples from WHI-P131-treated monkeys exhibited potent antileukemic activity against human ALL cells in vitro. To our knowledge, this is the first preclinical toxicity and pharmacokinetic study of a Janus kinase 3 inhibitor. Further development of WHI-P131 may provide the basis for new and effective treatment programs for relapsed ALL in clinical settings.

Animals↗
PubMed1999

Toxicity, biological activity, and pharmacokinetics of TXU (anti-CD7)-pokeweed antiviral protein in chimpanzees and adult patients infected with human immunodeficiency virus.

The purpose of the present study was to evaluate the toxicity and pharmacokinetics of TXU (anti-CD7)-pokeweed antiviral protein (PAP) in human immunodeficiency virus (HIV)-infected chimpanzees and adult patients. At a total dose of 100 microg/kg, TXU-PAP did not cause severe (grade >/= 3) toxicity in any of the four HIV type 1 (HIV-1)-infected or two healthy chimpanzees. The only side effects were a transient elevation of the liver enzyme alanine aminotransferase between days 2 and 14 without a concomitant rise in total bilirubin levels and a decrease in the serum albumin levels between days 1 and 5 without any concomitant weight gain or peripheral edema. TXU-PAP showed favorable pharmacokinetics in chimpanzees with a plasma elimination half-life of 5.1 to 12.0 h and a systemic clearance of 5.8 to 15.1 ml/h/kg. At 2 months after initiation of the TXU-PAP infusions, the HIV-1 burden was reduced to below-detection levels in three of the four chimpanzees, and in the remaining chimpanzee, the HIV burden was <500 RNA copies/ml at 2 weeks but returned to the pretreatment levels by 2 months. TXU-PAP was well tolerated by HIV-1-infected adult patients who received a single 5 microg/kg i.v. infusion of TXU-PAP. TXU-PAP showed very favorable pharmacokinetics in these patients with a relatively long plasma elimination half-life of 12.4 +/- 1.4 h, a mean residence time of 17.9 +/- 2.0 h, and a slow systemic clearance of 2.7 +/- 0.7 ml/h/kg. Concentrations of TXU-PAP required for effective inhibition of HIV-1 replication in preclinical models were achieved in HIV-1-infected patients at the 5 microg/kg dose level without any adverse reactions, and the mean value for AUC was 3059 +/- 721 ng. h/ml. The 1-h postinfusion plasma samples from TXU-PAP-treated patients showed potent anti-HIV activity in vitro and inhibited the replication of HIV in normal peripheral blood mononuclear cells (PBMCs) even at a 1:100 dilution. Although treatment with TXU-PAP at the 5 microg/kg dose level does not provide sustained therapeutic levels, it was capable of reducing the viral burden in six of six patients evaluated. To our knowledge, this is the first report of a clinical pharmacokinetics study of a PAP immunoconjugate in HIV-infected patients. The favorable long plasma elimination half-life of TXU-PAP in combination with its low toxicity provides the basis for further investigation of TXU-PAP as a potential anti-HIV agent.

Adult↗
PubMed1999

Treatment of therapy-refractory B-lineage acute lymphoblastic leukemia with an apoptosis-inducing CD19-directed tyrosine kinase inhibitor.

Seven children and eight adults with CD19+ B-lineage acute lymphoblastic leukemia, as well as one adult with chronic lymphocytic leukemia, were treated with the CD19 receptor-directed tyrosine kinase inhibitor B43-Genistein. All patients had failed previous chemotherapy regimens, and six patients had relapsed after bone marrow transplantation. B43-Genistein was administered as a 1-hour i.v. infusion at 0.1-0.32 mg/kg/day dose levels for 10 consecutive days or 3 consecutive days weekly for a total of nine doses. B43-Genistein was well tolerated by all patients with no life-threatening side effects. There were six episodes of grade 2-3 fever, two of which were clearly drug related, one episode each of grade 3 myalgia, grade 2 sinus tachycardia, and grade 2 vascular leak syndrome. There was one durable complete remission and two transient responses. Pharmacokinetic analyses in 12 patients revealed a plasma half-life of 20 +/- 5 h, mean residence time of 24 +/- 5 h, and a systemic clearance rate of 20 +/- 3 ml/h/kg. Moderate levels of human antimouse antibody (HAMA) ranging from 20-87 ng/ml were detected in the day 28 blood samples from three of nine cases examined. Treatment of these three HAMA-positive patients with a second course of B43-Genistein did not yield measurable immunoconjugate levels in the plasma, indicating that the administered B43-Genistein molecules were rapidly cleared from circulation due to the HAMA. On the basis of its acceptable toxicity profile and its ability to elicit objective responses at nontoxic dose levels, B43-Genistein may provide the basis for an effective treatment strategy for B-lineage acute lymphoblastic leukemia patients who have failed standard therapy.

Adolescent↗
PubMed1999

Rel/NF-kappaB represses bcl-2 transcription in pro-B lymphocytes.

The mechanisms controlling programmed cell death (PCD) during early B cell development are not well understood. Members of both the Bcl-2 family of apoptosis-related proteins and the nuclear factor-kappa B/Rel (NF-kappaB/Rel) family of transcription factors are expressed differentially during B cell development. To date, however, no direct interactions between these two families have been demonstrated. The FL5.12 cell line represents a model for progenitor B cell development. Such cells reproducibly undergo PCD upon IL-3 withdrawal. The signal to enter the apoptotic pathway is mediated by a shift in the ratio of Bcl-2:Bax. While bax levels remain constant, bcl-2 transcription rate, steady-state mRNA, and protein levels decrease. Analysis of the bcl-2 promoter reveals 3 kappaB sites functionally able to bind kappaB factors from FL5.12 nuclear extracts. Cotransfection studies demonstrate that NF-kappaB factors can repress bcl-2 transcription and that site-directed mutagenesis of the kappaB motifs abolishes this repression. These studies suggest that NF-kappaB mediates PCD in pro-B cells through transcriptional repression of the survival gene bcl-2, thus shifting the bcl-2:bax ratio in favor of death-promoting complexes.

Animals↗
PubMed1998

Enhancing effects of a mono-bromo substitution at the para position of the phenyl moiety on the metabolism and anti-HIV activity of d4T-phenyl methoxyalaninyl phosphate derivatives.

d4T-5'-[p-Bromophenyl methoxyalaninyl phosphate] (d4T-pBPMAP), a novel phenyl phosphate derivative of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) that has an enhanced ability to undergo hydrolysis due to the electron withdrawing properties of its single bromo substituent at the para-position of the phenyl moiety, was found to yield substantially more of the key metabolite alaninyl d4T monophosphate (A-d4T-MP) than the unsubstituted d4T-5'-phenyl methoxyalaninyl phosphate or para-methoxy substituted d4T-5'-phenyl methoxyalaninyl phosphate. d4T-pBPMAP was tested for its anti-HIV-1 activity in peripheral blood mononuclear cells (PBMNC) and thymidine kinase (TK)-deficient CEM T-cells. d4T-pBPMAP was 12.6-fold more potent than the parent compound d4T in inhibiting p24 production (IC50 values: 44 nM vs 556 nM) and 41.3-fold more potent than d4T in inhibiting the reverse transcriptase (RT) activity (IC50 values: 57 nM vs 2355 nM) in HIV-1-infected TK-deficient CEM cells. Similarly, d4T-pBPMAP was more potent than the unsubstituted or para-methoxy substituted phenyl methoxyalaninyl phosphate derivatives of d4T. d4T-pBPMAP did not exhibit any detectable cytotoxicity to PBMNC or CEM cells at concentrations as high as 10,000 nM. Notably, d4T-pBPMAP was capable of inhibiting the replication of a zidovudine (ZDV/AZT)-resistant HIV-1 strain as well as HIV-2 in PBMNC at nanomolar concentrations. To our knowledge, this is the first demonstration that the potency of the d4T-aryl-phosphate derivatives can be substantially enhanced by introducing a single para-bromo substituent in the aryl moiety.

Anti-HIV Agents↗
PubMed1998

A human lymphoid leukemia cell line with a V(D)J recombinase-mediated deletion of hprt.

Large deletions of exons 2 and 3 of the hprt gene are the most common type of hprt mutation in lymphocytes of newborn infants, and their frequency increases in cultured human T-lymphoid cells as a result of exposure to etoposide. Sequenced PCR products for these deletions are consistent with a V(D)J recombinase-mediated mechanism underlying their genesis. Herein, we describe the isolation and characterization of an etoposide-induced mutant CEM cell line that is clonal for a V(D)J recombinase-mediated exon 2 + 3 deletion. Human CCRF-CEM cells were exposed to 5 muM etoposide for 4 h, selected in 6-thioguanine, and an exon 2 + 3 deletion mutant was isolated through serial limiting dilution, using a PCR-based assay for detection of the exon 2 + 3 deletion. Untreated CEM cells and cells treated with 6-thioguanine alone were similarly subcultured. The exon 2 + 3 deletion-containing line was termed SJCEM808 and had a slightly longer doubling time than the control lines, tended to clump in suspension, and was characterized by cell membrane blebbing. Compared to the parent line, SJCEM808 had similar cytogenetic abnormalities, lower CD2, CD1, and CD10 expression, and negligible RAG-1 expression. However, RAG-1 expression was down-regulated in some untreated parental subclones following similar subculturing. The sequence of the exon 2 + 3 deletion mutation exhibited nucleotide insertions, and the breakpoints were adjacent to heptamer signal recognition sequences in intact hprt, consistent with a V(D)J recombinase-mediated mechanism underlying its genesis. There were no MLL gene or interlocus T-cell receptor (TCR) rearrangements. These results indicate that non-homologous recombination following etoposide treatment is neither necessarily accompanied by other large DNA rearrangements nor simply a pre-lethal event, and this cell line may serve as a useful tool for studying illegitimate V(D)J recombinase-mediated deletions.

Antigens, Surface↗