Biomedical subjects

C J Larsen

Publications and source records attributed to C J Larsen.

At least 109 records · Page 6Linked to original sources

PubMed1987

A single point mutation responsible for c-mos polymorphism in cancer patients.

A rare EcoRI restriction fragment length polymorphism (RFLP) in the 3' end of the human c-mos locus has been identified in DNA from patients with breast tumors, esophageal carcinomas and leukemias. Until now, this RFLP has not been found in normal populations, suggesting that its presence may reflect some cancer susceptibility. To characterize this RFLP, we have isolated both alleles of the c-mos locus from DNA of a breast cancer patient and determined the nucleotide sequence of the polymorphic region. Our results show that this RFLP is due to a single nucleotide substitution (T instead of C), resulting in the disappearance of EcoRI site.

Alleles↗

PubMed1987

[A variety of human autoantibodies recognizes in HeLa cells 2 proteins related to the TFIIIA factor of Xenopus laevis which regularizes the transcription of ribosomal 5S RNA].

Using the sera from two patients with autoimmune disorders, we have identified by immunoprecipitation of HeLa cell extracts two proteins with apparent molecular masses of 37 kDa (p 37) and 32 kDa (p 32). These proteins are associated with 5 S RNA. They are antigenetically related to Xenopus laevis 5 S RNA transcription factor TFIIIA, which is very abundant in early oocytes of this species. In contrast to what is observed in X. laevis oocytes, the TFIIIA-related proteins of HeLa cells are present in very small amounts. Our data suggest that proteins p 37 and p 32 are involved in the control of 5 S RNA transcription.

Animals↗

PubMed1986

A t(8;14)(q24;q11) translocation in a T-cell leukemia (L1-ALL) with c-myc and TcR-alpha chain locus rearrangements.

Cell lines established from T-cell leukemias have recently been reported to exhibit a chromosome translocation t(8;14) involving proto-oncogene c-myc and the gene of the T-cell receptor alpha-chain(TcR-alpha). In this work, we have studied a case of T-cell leukemia presenting a t(8;14)(q24;q11) translocation that was found in fresh leukemic cells taken during relapse, but was absent in cells collected at diagnosis. Hybridization analysis showed that the breakpoint on chromosome 8 was located 3' to the c-myc exon 3. A TcR-alpha-specific original probe (D14S7, Mathieu-Mahul et al., 1985) revealed two differently rearranged patterns in DNA from leukemic cells obtained at diagnosis and during relapse. In contrast, the rearranged TcR-beta-gene DNA pattern did not change during the course of the disease, indicating that leukemic cells were clonally related. These data indicate that the chromosome breakpoint in 14q11 is situated in the TcR-alpha locus. These results suggest that translocations t(8;14) involving TcR-alpha and c-myc genes in T-cell malignancies are analogous to variant t(2;8) and t(8;22) translocations observed in Burkitt lymphoma. They also establish that the same types of molecular rearrangements due to a t(8;14)(q24;q11) translocation, at first described in T-cell lines established in culture, also exist in vivo and may play a role in the evolution of the leukemic process.

Child↗

PubMed1986

Characterization of the topoisomerase II-induced cleavage sites in the c-myc proto-oncogene. In vitro stimulation by the antitumoral intercalating drug mAMSA.

In an attempt to get an insight into the activity of mAMSA (a DNA topoisomerase II-mediated drug) on the human proto-oncogene c-myc, an in vitro system consisting of purified calf thymus DNA topoisomerase II and a c-myc DNA inserted in lambda phage was utilized. The occurrence of discrete bands, detected by hybridization of Southern blots with appropriate c-myc probes, indicated the presence of cleavage sites in the sole presence of DNA topoisomerase II. The band intensity increased in the presence of mAMSA, while no significant difference occurred in the cleavage pattern. The location of the cleavage sites along the c-myc locus revealed a striking correspondence with that of some DNase hypersensitive sites. These results indicate that DNA topoisomerase II is most certainly implicated in the mAMSA activity and that the drug stimulates the topoisomerase II cleaving activity at specific sites, which may be involved in the biological activity of the drug.

Amsacrine↗

PubMed1986

The in vitro involvement of topoisomerase II in the activity of aza-ellipticine analogues is not correlated with drug activity on isolated nuclei.

Aza-ellipticines are DNA intercalative ellipticine analogues with antitumor activity that induce protein-linked DNA breaks in NIH 3T3 cells in culture. The effects of two aza-ellipticine congeners (BD-40 and BR-76) on the activity of purified Calf Thymus type II topoisomerase were studied using pUC13 DNA as substrate. DNA cleavage was stimulated by both molecules at those doses required for inducing lethal effects in cells (DE5O). This effect was reversed by high salt treatment, indicating that it was actually mediated by Topo II. Mapping of cleavage sites on linearized and 3' end-labelled pUC13 DNA showed that ellipticine and aza-ellipticines stimulated the same sites, which differed from those stimulated by m-AMSA. Decatenating activity of Topo II on Trypanosoma cruzi kDNA was both inhibited by ellipticine and BD-40 at concentrations much higher than DE50 concentrations. Activity of aza-ellipticines was also investigated on isolated nuclei. Unlike ellipticine which promoted DNA-breaking activity, BD-40 and BR-76 were repeatedly inactive. Prior treatment of DNA by Proteinase K did not reveal hidden breaks which are formed in intact cells treated with BD-40 (Vilarem et al., 1984, Nucleic Ac. Res. 12, 8653). Concordant with these data, BD-40 did not impair DNA-synthetic activity in isolated nuclei, while Ellipticine largely decreased it. These results indicate that lesions induced in DNA by Aza-ellipticines are mediated by Topo II. The absence of effect of these drugs on isolated nuclei compared to that of Ellipticine may be due to some specific features of the association between Topo II and Aza-ellipticines or reflect a bioactivation step as a prerequisite for in vivo activity.

Alkaloids↗

PubMed1986

In vivo stimulation by antitumor drugs of the topoisomerase II induced cleavage sites in c-myc protooncogene.

Several antitumor drugs including DNA intercalative and non intercalative agents induce in vitro and in vivo double-stranded DNA breaks by stabilization of a topoisomerase II-DNA complex. In order to locate cleavage sites in an actively transcribed oncogene, N417 cells, originating from a human small cell lung carcinoma and containing 45-50 copies of c-myc oncogene, were treated with mAMSA, 9 hydroxyellipticine and VM 26. The presence of DNA lesions in c-myc was investigated by Southern blot hybridization with a human c-myc probe. In addition to normal bands, DNA patterns of drug treated-cells revealed the presence of new bands most likely corresponding to topoisomerase II-mediated cleavage as these bands were not found in untreated control DNA and in DNA treated with oAMSA, a biologically inactive stereoisomer of mAMSA. Major cleavage sites induced by drugs in the N417 cell c-myc locus were located in the 5' end of the c-myc exon 1 closely to some DNAse I hypersensitive sites which are assumed to reflect an activity of the gene. Therefore our data suggest that TopoII-mediated drug activity correlates with gene activity.

Aminoacridines↗

PubMed1986

Detection of T cell receptor alpha chain locus rearrangements in human antigen-specific T lymphocyte clones using a DNA probe specific for chromosome region 14q11.

Human cloned antigen-specific T lymphocytes were used to characterize DNA rearrangements using a cDNA probe from the T cell receptor (TCR)-alpha chain gene. Rearranged patterns were detected in some T cell clones, confirming that normal mature T cells are rearranged in TCR-alpha locus. Similarly, rearranged DNA patterns were found in T cell clones from the same panel, using a DNA probe (clone K-40, [1]) isolated from chromosome 14 (14q11), where the TCR-alpha locus has been mapped. These results suggest that this genomic DNA clone is located within the TCR-alpha chain locus.

Chromosome Mapping↗

PubMed1986

[Translocation t(1; 14) and rearrangement of the gene for the alpha chain of the T-cell receptor in acute T-lymphoblastic leukemia].

A t(1; 14) (p32; q11) translocation has been found by the cytogenetic study of a T cell acute lymphoblastic leukemia patient. Molecular hybridization with a probe (D14S7) which recognizes a fragment of the alpha chain T receptor gene has shown a rearranged EcoRI 11.0 kb band in addition to the germline 3 and 8 kb bands. This translocation infrequently described until now is a new example of an alpha chain gene rearrangement in T cell malignancy. Moreover the breakpoint on chromosome 1 may also involve the newly isolated protooncogene, L-myc, normally localized on the chromosomal band 1 p32.

Adolescent↗

PubMed1985

Molecular cloning of a DNA fragment from human chromosome 14(14q11) involved in T-cell malignancies.

To isolate DNA segments specific to chromosome band 14q11, which has been implicated in a number of human T-cell malignancies, a genomic DNA library was prepared from a variant cell subline of the human lymphoblastic KE37 cell line. This subline (KE37-R) bears a t(8;14) (q24;q11) translocation, and the breakpoint on the resulting chromosome 8q+ has been located at the 3' end of the third c-myc exon. Three molecular clones were isolated by screening the library with a c-myc exon 3 probe, and one of them (lambda K40) was analyzed in detail. It contains a 15-kb insert consisting of 4.5 kb of sequence from chromosome 8 (e.g., downstream of c-myc exon 3) and sequences from chromosome 14. The origin of these latter sequences was established by hybridizing DNA from chromosomes sorted by flow cytometry to a lambda K40 subclone containing only chromosome 14 presumptive sequences and by Southern blot analysis of rodent X human somatic hybrid cell DNA with the same probe. No cross-hybridization was found between the lambda K40 clone and a cDNA clone for the alpha chain T-cell receptor gene which is also located in 14q11. A preliminary survey of DNAs from human T-cell malignancies with a probe corresponding to chromosome 14 sequences of lambda K40 clone revealed for some of them restriction patterns different from those of the germ line DNA. The fact that the rearrangement observed in a leukemic patient was not found in DNA from lymphocytes obtained during remission excluded any polymorphism.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosomes, Human, 13-15↗

PubMed1985

Presence of circulating antibodies against gag-gene MuLV proteins in patients with autoimmune connective tissue disorders.

An immunoblotting procedure using viral proteins from purified murine sarcoma virus or MSV-(MLV) has been developed to characterize antiviral antibodies in sera from patients with autoimmune connective tissue disorders. Fifty-eight sera with anti-Sm, anti-RNP, anti-SS-B (La), and other undefined specificities were found to react with several major viral polypeptide bands. Most of them corresponded to gag-gene-encoded products: pr65gag, p40gag, p30, p15, p12 and p10. Other bands with molecular weights averaging 90K, 60K, 45K, and 28K were recognized by a few sera. Immunological specificity of the reaction was assessed by reproducing the tests with IgG purified from sera and from corresponding F(ab')2 fragments. Moreover, the specificity of the reaction with gag proteins was confirmed by repeating the tests with p30 and p15 prior purified by immunoprecipitation with anti-p30 and anti-p15 goat sera. Furthermore, the gag polypeptides were recognized by human sera by replacing MSV-(MLV) by three other murine retroviruses of different origin. An indirect confirmation of these results was obtained by applying this method to sera of MRL lpr/lpr mice which develop an autoimmune syndrome comparable to that of human systemic lupus erythematosus. In agreement with previously published results (C. Rordorf, C. Gambke, and J. Gordon (1983), J. Immunol. Methods 59, 105-112), we found that anti-gag-gene antibodies were present in the sera of individual mice. Patterns of reactivity were found to vary with the age of the animals. No retroviral polypeptide was significantly detected in the great majority (80%) of sera from normal donors. However, 5 out of 25 sera showed faint bands although to a lesser extent than pathological sera. These five sera also reacted with HeLa cell purified HnRNPs, suggesting that their normal status should be reconsidered.

Antibodies, Viral↗

PubMed1985

Human proto-oncogene c-mos maps to 8q11.

The c-mos proto-oncogene is the cellular counterpart of the viral oncogene v-mos isolated from Moloney murine sarcoma virus. The c-mos gene locus has previously been assigned to human chromosome 8. By both in situ hybridization and molecular hydridization to sorted chromosome DNA (using a c-mos probe) we have localized the c-mos gene to band 8q11. This regional localization is at variance with the one previously reported at 8q22 and may explain why no rearrangement of c-mos has been found in acute leukaemia with the chromosomal translocation t(8;21)(q22;q22).

Animals↗

PubMed1985

An EcoRI restriction fragment length polymorphism (RFLP) in the human c-erb A locus.

An EcoRI restriction fragment length polymorphism (RFLP) was detected in the 3' end of the locus of the c-erb-A proto-oncogene. The frequency of the rarer allele was around 3.0% in a normal population of 107 unrelated individuals. This frequency did not significantly differ in DNA samples from patients with breast tumors or acute leukemias.

Alpharetrovirus↗

PubMed1985

PSL, an S phase-related p55 nuclear antigen, associates transiently with chromatin.

An S phase-related nuclear 55K antigen, also designated PSL, has been characterized in various mammalian cells, using a human serum from a patient with autoimmune disorders (Barque et al., EMBO j 2 (1983) 743). In this report, we show by immunoelectron microscopy that the p55 protein associates in situ with the chromatin of rat hepatocytes. This association is a transient one, as indirect immunofluorescence studies show that PSL does not bind to individualized metaphase chromosomes. Furthermore, immunoprecipitation tests indicate that the majority of PSL is in the non-chromosomal cell fraction. These results suggest that this nuclear antigen is directly involved in the DNA replication process.

Animals↗

PubMed1985

Presence of an allelic EcoRI restriction fragment of the c-mos locus in leukocyte and tumor cell DNAs of breast cancer patients.

Structure of the human c-mos protooncogene in DNAs from breast tumors, leukemic cells, and lymphocytes from normal individuals was analyzed by restriction enzyme digestion and Southern blot. In 6 of 75 breast tumor DNAs, we found an EcoRI 5-kilobase extra band hybridizing with a human c-mos probe containing all of the sequences homologous to v-mos oncogene. This band was also found in lymphocyte DNA from 3 of these patients, indicating a restriction fragment length polymorphism. This polymorphism was not found in a series of 69 lymphocyte DNAs from the unaffected population. Moreover, 1 of 73 leukemic cell DNAs exhibited the 5-kilobase band. These results indicate that this rare polymorphism is significantly more frequently found in patients with breast cancer than in the rest of the population (P less than 0.05, by a chi 2 test with Yates correction.

Alleles↗

PubMed1985

[Rearrangement of the proto-oncogene c-myc in fresh cells from Burkitt's leukemia (L3)].

The presence of a rearrangement of the proto-oncogene c-myc was investigated in the DNA of fresh cells isolated from the blood of two patients with Burkitt's leukemia (L3), and from the node biopsy of a patient with Burkitt's lymphoma. Both samples from the L3 leukemia patients had the characteristic t(8;14) translocation, while the lymphoma specimen presented no abnormality of chromosome 8. Only one of the leukemic DNA's presented a rearranged c-myc pattern, with the breakpoint region located between the first and the second exon. The c-myc pattern of the two other patients appeared normal. The finding of a rearranged c-myc oncogene in fresh cells from a Burkitt's leukemia is direct evidence for the implication of c-myc in the disease whereas most of the rearrangements previously described have been found in cell lines established in culture.

Base Sequence↗

PubMed1985

[Rearrangement of the c-myc proto-oncogene locus in a cell line of T-lymphoblastic origin].

A molecular rearrangement of the proto-oncogene c-myc located downstream of the exon III 3' end has been found in a cell line derived from KE37 cell line established from an acute T-lymphoblastic leukemia. This rearrangement resulted from a chromosomal translocation t(8; 14)(q24; q11). Since the 14q11 chromosomal band has been found to be involved in several T-cell leukemias and lymphomas, the importance of the rearrangement of c-myc discovered in the KE37 cell line lies in the possibility of analyzing chromosome 14 DNA near the breakpoint involved in the translocation.

Cell Line↗

PubMed1985

Differential effects of ellipticine and aza-analogue derivatives on cell cycle progression and survival of BALB/c 3T3 cells released from serum starvation or thymidine double block.

10-[Diethylaminopropylamino]-6-methyl-5H-pyrido[3',4':4,5] pyrrolo[2,3-g]isoquinoline (BD-40) (NSC-327471D) is an aza-ellipticine derivative with a promising antitumor activity (M. Marty, C. Jasmin, P. Pouillard, C. Gisselbrecht, G. Gouvenia, and H. Magdalainat, 17th Annual Meeting of the American Society of Clinical Oncology, C-108, 1981) and less toxicity than ellipticine. We have compared the effects of ellipticine, several of its analogues, and two aza-analogue ellipticine derivatives (BD-40 and BR-1376) on cell cycle progression of BALB/c 3T3 mouse cells under different growth conditions. Both drug series were found to stop cell growth and block cells in G2 phase in exponentially growing cultures and cultures released from a thymidine double block. Long-term viability of these cells was completely suppressed after a short exposure to the drugs. In contrast, while ellipticine and its derivatives caused identical effects in cells recovering from serum starvation, BD-40 and BR-1376 did not block cells in G2 phase and did not prevent the completion of the first division round occurring after serum addition to quiescent cells. This transient refractory state was accompanied by a total conservation of long-term viability of these cells at least for the next 6 h following serum and drug addition. This lack of effect was not related to an impaired drug uptake by cells recovering from serum starvation or by a dramatic change in drug distribution inside the cells. These results indicate that the nitrogen substitution in the ellipticine heterocycle is an important if not unique feature for the particular effect of the aza-analogues of ellipticine. Furthermore, they suggest that, in contrast to ellipticine derivatives, these compounds require an activation step before exhibiting cytotoxicity.

Alkaloids↗

PubMed1984

BD-40, an ellipticine-related DNA intercalative agent induces DNA-protein bridges in vivo.

The ability of BD-40, a DNA intercalative Ellipticine analogue, to induce DNA protein-cross links (DPLs) in mammalian cell DNA was studied by measuring DNA extractability with 0.5M KCl. Like Ellipticine, BD-40 was found to decrease the extractability of DNA, indicating the presence of DPLs, at inhibitory doses as well as at cytotoxic doses. Further analysis by alkaline sucrose gradient sedimentation of BD-40-induced DLPs indicated that the apparent size of DNA was not modified in comparison to that of control cell DNA. Abolition of DPLs by a prior proteinase K treatment of nuclear lysates resulted in DNA size reduction revealing the existence of hidden DNA strand breaks. In contrast, no proteolytic treatment was required to obtain a similar size reduction of DNA from Ellipticine-treated cells. These data suggest that BD-40 induced DPLs involve NaDodSO4+alkali-resistant DNA strand-protein bridges which maintain cohesiveness of adjacent DNA termini. Thus, BD-40 appears to be different from other DPL-inducing intercalative agents which have not been reported to induce DNA-protein bridges.

Animals↗