Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events.

ICRF-193, a novel noncleavable, complex-stabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T. Miki, T. Narita, R. Yui, S. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Cancer Res. 51:4909-4916). With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated. Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes. In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by ICRF-193. Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases. In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of cdc2 kinase, spindle apparatus reorganization and disassembly and reassembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase. Cells then continued through further cell cycle rounds, becoming polyploid and losing viability. This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast. The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes. In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes.

Rearrangements on chromosome 11q23 in hematopoietic tumor-associated t(11;14) and t(11;19) translocations.

We previously demonstrated that the breakpoint of t(11;14)(q23;q32) in the RC-K8 B cell lymphoma cell line lies between CD3 and THY1/ETS1 on chromosome 11q23, and we cloned this region and named it the rck locus. Pulsed-field gel electrophoresis showed that the rck probe B (distal to the breakpoint) and the porphobilinogen deaminase (PBGD) probe detect the same germ line band and also the same rearranged band when DNA from RC-K8 cells was digested with NotI enzyme. Furthermore, Southern blot analysis with somatic cell hybrids showed that the PBGD gene moved to the 14q+chromosome, which confirmed PBGD to be more distal to the centromere than the rck locus. These data allowed us to construct the following order of genes: 11 cen-q23-CD3-rck-PBGD-THY1/ETS1. In this study, three infantile leukemia cell lines with t(11;19)(q23;p13) translocation were also analyzed by pulsed-field gel electrophoresis. CD3D probe detected the rearranged bands in DNA from two of them after digestion with NotI and SacII enzymes, demonstrating that the breakpoints of both cell lines were estimated to be within 360 kilobases of CD3D.

Inhibition of intracellular topoisomerase II by antitumor bis(2,6-dioxopiperazine) derivatives: mode of cell growth inhibition distinct from that of cleavable complex-forming type inhibitors.

In the accompanying paper (K. Tanabe, Y. Ikegami, R. Ishida, and T. Andoh, Cancer Res., 51: 4903-4908, 1991), we showed that ICRF-154 and -193, dioxopiperazine derivatives, inhibited the activity of purified topoisomerase II, without formation of a cleavable DNA-protein complex. In order to see whether ICRF-154 and ICRF-193 affect cellular topoisomerase II in situ or not, we examined the effect of these drugs on etoposide (VP-16)-induced, topoisomerase II-mediated DNA breaks in RPMI 8402 cells by alkaline sedimentation analysis. When RPMI 8402 cells were exposed to VP-16 in the presence of ICRF-154 or ICRF-193 for 1 h, VP-16-induced DNA strand breaks were greatly inhibited by both ICRF compounds. In parallel with this observation, VP-16-induced growth inhibition was also reversed by ICRF-193. Exposure of cells to ICRF-154 resulted in a progressive accumulation of cells with 4C DNA content. Although mitotic index did not significantly increase, mitotic abnormalities were seen in cells exposed to ICRF-193 or ICRF-154: all mitotic cells exhibited early mitotic figures with fewer condensed and entangled chromosomes. The most sensitive phase of the cell cycle to ICRF-154 was the G2-M. ICRF-154 did not affect the spindle formation. However, abnormally oriented spindles were observed in drug-treated cells in parallel with the appearance of multinucleated cells. The results suggest that ICRF-154 and -193 inhibit topoisomerase II activity in RPMI 8402 cells, and this effect resulted in the appearance of cells in G2 and early M phase with fewer condensed and entangled chromosomes and of cells with multilobed nuclei.

Antigenic phenotype and biological characteristics of two distinct sublines derived from a small cell lung carcinoma cell line.

Two sublines, SCLC-MOA1 (MOA1) and SCLC-MOA2 (MOA2), were established from the SCLC-MO cell line, which was originally derived from an oat cell type of small cell lung carcinoma (SCLC). SCLC-MO showed typical culture morphology of SCLC, growing as tightly packed floating aggregates, while both MOA1 and MOA2 grew as a monolayer. MOA2 showed markedly shorter culture doubling time and higher colony forming efficiency than SCLC-MO and MOA1. When transplanted into nude mice, both SCLC-MO and MOA1 showed intermediate cell type histology, while MOA2 showed a picture of large cell carcinoma as non-SCLC. As for biomarkers, SCLC-MO showed a transitional state between the classic and the variant types, while MOA1 was the variant type. In contrast, MOA2 lost the biomarker characteristic of SCLC, showing rather non-SCLC type. SCLC-MO expressed NE-150 neuroendocrine antigen, but lacked PE-35 panepithelial antigen which is generally present on SCLC. It lacked also OE-130 epithelial antigen which is generally absent from SCLC. Thus, the phenotype was NE-150+/PE-35-/OE-130-, which was different from the major phenotype of SCLC, NE-150+/PE-35+/OE-130-. MOA1 was weakly positive for PE-35, showing NE-150+/PE-35 +/- /OE-130-, while MOA2 was positive for OE-130, but lost NE-150, i.e., NE-150-/PE-35+/OE-130+, showing a non-SCLC phenotype. Thus, a good concordance was observed between the antigenic phenotype and the biological characteristics of these SCLC lines. The results altogether suggested that a part of large cell carcinoma in the tumor of the patient may be derived from SCLC. Karyotype analysis showed that there were several marker chromosomes including deletion of chromosome 3p shared by these three cell lines, supporting the belief that MOA1 and MOA2 originated from SCLC-MO. Southern blot analysis showed the amplification of the L-myc related gene, probably rearranged L-myc, in the primary SCLC tumor as well as in SCLC-MO and MOA1. Northern blot analysis showed the 2.2-kilobase transcripts hybridized with a L-myc probe were observed in SCLC-MO and MOA1, but not in MOA2. In contrast, the c-myc transcript was detected only in MOA2. The activity of the myc gene family may contribute to certain biological characteristics of SCLC.

Gene rearrangement and overexpression of PRAD1 in lymphoid malignancy with t(11;14)(q13;q32) translocation.

The proto-oncogene PRAD1 (parathyroid adenoma 1) on chromosome 11q13 was found to be overexpressed in all five B-cell lines with t(11;14)(q13;q32) translocation tested. One B-cell lymphoma and four myeloma cell lines with this translocation demonstrated more than 10-fold overexpression as determined by Northern blot analysis, when compared with normal lymphoid tissues such as thymus, spleen and lymph node. Hematopoietic cell lines without the translocation were also examined, but none of these demonstrated the overexpression, confirming that overexpression of the PRAD1 gene is associated with t(11;14) translocation. A truncated form of mRNA was seen in one of five cell lines with the translocation, SP-49. Hybridization with different regions of the PRAD1 cDNA revealed that the truncated form of mRNA retained the coding region but had lost the 3' untranslated region. Southern blot analysis demonstrated a gene rearrangement in this SP-49 cell line. To study the genetic alteration responsible for the truncated form of mRNA in this cell line, the rearranged allele as well as the germline allele were cloned. The restriction map revealed that the rearranged portion was at the 3' end of the PRAD1 gene, eliminating the mRNA-destabilizing signal AUUUA. Human-rodent hybrid cell analysis demonstrated that the region introduced 3' of PRAD1 was derived from chromosome 11, suggesting that the PRAD1 gene region is deleted at the 3' end. Over-expression of the PRAD1 gene in association with t(11;14)(q13;q32) translocation suggested that in these cases the regulation of PRAD1 was altered by the juxtaposed gene, most likely the immunoglobulin heavy-chain gene from chromosome 14.

Cytogenetic studies on rat thymic lymphomas induced by N-propyl-N-nitrosourea.

N-Propyl-N-nitrosourea (PNU) was proved to be a strong leukemogen, which induces myelogenous leukemia or thymic lymphoma in rats. BUF/Mna rats and F344 rats were the strain most susceptible to thymic lymphomagenic activity of PNU. In addition, F1 rats between BUF/Mna and WKY rats were also susceptible to PNU-lymphomagenic activity. In the present experiment, karyotypes of 31 thymic lymphomas induced by PNU in BUF/Mna rats and in F1 rats between BUF/Mna and WKY rats were analysed for chromosomal abnormalities. Although no specific chromosomal abnormalities were observed throughout all lymphomas, del(11q) and dup(2q) were observed frequently in BUF/Mna rat lymphomas. Breakpoints and/or fusion-points were frequently observed in chromosome 11, followed by chromosomes 2, 5 and 6. Trisomy of chromosome 7, on which c-myc oncogene is mapped, was observed in seven cases, and monosomy of chromosomes 12, 18, 19, 20 and X was seen in seven or eight cases each, though these changes were generally observed in minor cell population in each case.

Cytogenetic characterization of a T-cell line, ATN-1, derived from adult T-cell leukemia cells.

A T-cell line, ATN-1, was established by culturing peripheral blood mononuclear cells derived from a patient with adult T-cell leukemia/lymphoma (ATL/L). Identities of the patterns of chromosomal abnormalities, cell surface phenotypes, morphologic findings, rearrangement patterns of T-cell receptor beta chain gene, and an integration site of human T-cell leukemia virus I proviral genome indicated that ATN-1 was derived from original leukemic cells. Both ATN-1 and the original leukemic cells showed a variety of patterns of chromosomal abnormalities that include 3q-, 6q-, rearrangements involving 2q31, 7q11.2, 8q11, 8q24, 19p13.3, and also 14q11 and 14q32, where genes for the T-cell receptor alpha chain and the immunoglobulin heavy chain are located. Availability of a genuine ATL/L cell line with these chromosomal abnormalities may greatly facilitate the biologic analysis of ATL/L.

Sister-chromatid exchanges (SCEs), cell survival and mutation in HeLa s3 cells with different sensitivity to alkylating agents; evidence that SCE induction and cell survival or mutation induction are dissociable.

We previously isolated N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistant cells, MR from HeLa S3 Mer- cells. In the present study, we have isolated 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)-resistant cells, ACr. The MR cells had only a little O6-methylguanine-DNA methyltransferase (MT) activity, while the ACr cells had increased MT activity and also became resistant to the cytotoxic effect of MNNG. We compared the induction of sister-chromatid exchanges (SCEs), cell survival and mutation in these HeLa S3 cells with different sensitivity to MNNG. The ACr cells were much more resistant than the parental HeLa S3 Mer- cells to cytotoxicity, mutagenicity and SCE induction by MNNG, showing a positive correlation between SCE induction and cell killing or mutation. In contrast, this positive relationship was not observed between HeLa S3 Mer- and MR cells. These results suggest that O6-methylguanine (O6-MeG) is involved in the induction of the biological effects of MNNG such as cytotoxicity, mutagenicity and SCEs, and also indicate that SCE induction does not always correlate with cell killing and mutation.

Expression of proto-oncogenes and tumor suppressor genes in in vitro cell lines derived from a thymus, thymoma, and malignant thymoma of rats.

To analyze the processes of the development of thymoma and malignant thymoma from normal thymic epithelial cells, we have examined the expression of 15 proto-oncogenes and tumor suppressor genes in seven in vitro epithelial cell lines established from a normal thymus (TuD1-1, TuD1-3, and TuD1-5), thymoma (TaD1-3 and TaD1-8), and malignant thymoma (MTHC-1 and MTHC-3) of rats. Northern blot analysis indicated that most of these genes examined were transcribed at similar levels. However, higher levels of transcription of epidermal growth factor receptor (EGFR) were observed in TuD1-1, TuD1-3, TuD1-5, TaD1-3, and TaD1-8 cells than in MTHC-1 and MTHC-3 cells. Conversely, four of the former five cell lines showed no TGF-beta transcription while the latter two cell lines had high levels of its expression. In addition, c-fos proto-oncogene was highly expressed in TuD1-5 cells, which showed the fastest growth rate among the seven cell lines. These results denote that some molecular changes in the regulation of gene expression occurred in the processes of malignant transformation of thymic epithelial cells.

A scanning electron microscopic study of chromosomes and nuclei.

The fine structure of metaphase chromosomes and nuclei were studied by scanning electron microscopy. A coiled-coil structure of chromosomes was suggested by hypotonically unravelled chromosomes observed under light microscope. But chromosome preparations made for the light microscopic studies were not adequate for detailed examination with scanning electron microscope. Surface-spread chromosomes revealed that they were composed of nodular, twisted looping fibers of about 300 A in diameter. Surface-spread nuclei were also composed of fibers identical to the chromosome fibers.

Scanning electron microscopy of Giemsa-stained chromosomes and surface-spread chromosomes.

Giemsa-stained chromosomes as prepared for light microscopy, and including G-banded, C-banded, and FPG-stained chromosomes, were examined by scanning electron microscopy. Although suitable for light microscopy, these chromosomes were too flat for a close examination of their fine structure by scanning electron microscopy. The surface of Giemsa-positive regions was rough and bright, whereas that of unstained or poorly stained regions was smoother and less bright. Giemsa-staining, therefore, seems to produce the bulkiness of the chromosomes. On topographical examination by scanning electron microscopy, the transparent chromosomes as observed with the light microscope proved to be "footprints". Stereographical examinations of surface-spread chromosomes showed that minimally stretched chromosomes were composed of a mass of nodular and twisted looping fibers with an average diameter of about 300 A. The substructure of these chromosome fibers was not determined. The kinetochore region was discernible as a constriction in the mass of the chromosome fibers, and was distinguishable from gaps by the presence of several chromosome fibers parallel to the axis of the chromatid. The organization of the chromosome fibers, however, was disordered rather than regular.

Differences in induction of sister chromatid exchanges in MNNG-resistant HeLa S3 Mer- cell lines.

All N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistant (MR) cell lines of HeLa S3 Mer- cells tested were found to be defective in O6-methylguanine-DNA methyltransferase and more sensitive than HeLa Mer+ cells to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-1-nitroso-ure a, like the parental HeLa S3 Mer- cells. MR10-1 and MR1-3 cells were also highly resistant to induction of sister chromatid exchanges (SCEs) by MNNG, while MR6, MR8 and MR11 cells were slightly resistant to SCE induction, MR10-1 and MR1-3 cells showed higher spontaneous mutation rates than the parental cells. These results indicated that there are at least two types of MR cells. All MR cells were more sensitive than HeLa S3 Mer- cells to 6-hydroxyamino purine.

Topographic examination of sister chromatid differential staining by Nomarski interference microscopy and scanning electron microscopy.

BrdU-substituted Chinese hamster chromosomes were treated with a hog Na2HPO4 solution and stained with Giemsa to produce sister chromatid differential staining (SCD). The process of SCD was examined with the Nomarski differential interference microscope and the scanning electron microscope. After the Na2HPO4 treatment alone, unifilarly BrdU-substituted (TB) chromatids appeared somewhat more severely collapsed than the bifilarly substituted (BB) chromatids. Subsequent Giemsa staining, however, brought about pronounced piling up of the Giemsa dye on the TB-chromatids but not on the BB-ones, causing highly distinct differential Giemsa staining as well as a marked differentiation in surface topography between the sister chromatids. Removal of the Giemsa dye from the differentially Giemsa stained chromosomes resulted in a disappearance of such a pronounced topographic differentiation.

Effect of ATP on the release of hsp 70 and hsp 40 from the nucleus in heat-shocked HeLa cells.

We have recently found a novel 40-kDa heat-shock protein (hsp 40) in mammalian and avian cells and reported that the N-terminal amino acid sequence of mammalian hsp 40 has homology with the bacterial DnaJ heat-shock protein. Also, hsp 40 has been shown to be translocated from the cytoplasm into the nuclei/nucleoli by heat shock and colocalized with hsc 70 (p73) in the nucleoli of exactly the same cells. We here investigated the effect of ATP on the release of hsp 70 (both constitutive p73 and inducible p72) and hsp 40 from the nuclei/nucleoli of heat-shocked HeLa cells which were permeabilized with Nonidet-P40 using immunofluorescence and immunoblotting. Hsp 70 in the nucleoli was released by the addition of ATP but not by ADP, GTP, nonhydrolyzable ATP, nor high salt buffer. In contrast, hsp 40 was not released from the nucleoli with any of these treatments or any combination of these treatments. Thus, hsp 40 might dissociate spontaneously from the nucleoli after hsp 70 has been released in an ATP-dependent manner. Using cell fractionation methods, we showed that while the majority of hsp 40 is localized in the cytoplasm, a small portion of it is located in the microsome fraction in non-heat-shocked control cells and in cells which recovered from heat shock.

Human monoclonal antibody reactive to stomach cancer produced by mouse-human hybridoma technique.

Production of human monoclonal antibodies reactive to stomach cancer was attempted by the hybridoma technique using splenic lymphocytes from stomach cancer patients. The parental cells used were NS-1 mouse myeloma line and three human lines including RPMI-1788 6TGR, which was established in our laboratories. Ten mouse-human and two human-human (from the fusion with RPMI-1788 6TGR) hybridomas have been producing IgM antibody for over 18 months, and all the heterohybridomas yielded ascites when transplanted into nude mice. Four antibodies produced by the heterohybridomas were selected and analyzed. These 4 antibodies, 3F6, 4A10, 3H5 and 1F9, reacted predominantly to cytoplasmic antigens of stomach and other epithelial cancer lines. The reactivity against human tumors transplantable in nude mice showed that all antibodies but 3F6 were reactive with stomach and lung cancers. Smears prepared from normal and cancer tissues were also tested, and these 4 antibodies showed positive reactions not only to stomach cancer, but also to normal stomach and colon. The reactivity against fetal tissues demonstrated that 3H5 antibody was reactive with epithelium of the stomach, and 1F9 antibody was positive with epithelium of the respiratory tract and bile duct, but the other two were negative. Thus, the serological analysis showed that the antigens detected are not tumor-specific, but are differentiation antigens. Chromosome analysis of these 4 mouse-human hybridomas and another one, which seems to produce an antibody against keratin, showed that three retained human chromosome 14 on which immunoglobulin heavy chain (Ig H) gene is located, but two did not. Southern blot analysis, however, revealed that all 5 hybridomas had a human Ig H gene.

Assignment of the gene for human DNA polymerase beta (POLB) to chromosome 8.

A cloned cDNA segment of 442 bp which contains the region coding the N-terminal sequence of rat DNA polymerase beta polypeptide was used as a probe in Southern hybridization analysis to identify the gene for this enzyme. This probe hybridized with DNA from mouse and human in addition to rat DNA, and the BamHI fragment containing DNA polymerase beta gene sequence was unique in size in each species: 15.8 kb for rat DNA, 8.7 kb for mouse DNA and 10.1 kb for human DNA. DNA extracted from a panel of 12 independent human-mouse somatic cell hybrids was analyzed by Southern blot hybridization with the cloned cDNA probe. The results indicate that the gene for the human DNA polymerase beta sequence (POLB) is located on chromosome 8.

Gene encoding human p250 T-cell activation antigen maps to human chromosome 11.

Human p250 T-cell activation antigen detected by a monoclonal antibody, B1.19.2, is a single peptide antigen with a molecular weight of 250 kilodaltons and is classified serologically into cluster of differentiation, CDw26. Concordance between the presence of human chromosome 11 and the reactivity with B1.19.2 was demonstrated by chromosomal analysis of 23 clones derived from three hybrid series obtained from the fusion of human activated lymphocytes or T-cell chronic lymphocytic leukemia cells and BW5147 mouse T-cell leukemic cells. The results indicated that the presence of chromosome 11 was essential for the expression of p250 T-cell activation antigen. Moreover, the gene for this antigen was assigned to chromosome 11pter----p11.2 by analysis of the hybrid clones retaining the translocated chromosome of 11.

Assignment of gene coding human T-cell differentiation antigen, Tp120, to chromosome 11.

A pan T-cell antigen with a molecular weight of 120 kilodaltons (kd) is recognized by a monoclonal antibody, Tp120, produced in our laboratories. Two hybrid clones reactive with this Tp120 antibody were established from the fusion between concanavalin A-stimulated human peripheral blood lymphocytes and hypoxanthine-guanine phosphoribosyltransferase-deficient mouse T cell leukemia, BW5147. These two clones were also positive with two other antibodies, 12.1 and T12, both of which detect 120kd pan T-cell antigen. Karyotype analysis showed that one clone retained human chromosomes 6, 7pq-, and 11, and the other maintained chromosomes 11 and 21. As soon as both of these clones lost the chromosome 11, the expression of Tp120 became negative. The presence of human chromosome 11 was confirmed by the isozyme analysis of lactate dehydrogenase-A. The results indicated that the presence of chromosome 11 was essential for expression of 120kd pan T-cell antigen.

Comparative study on E6 and E7 genes of some cutaneous and genital papillomaviruses of human origin for their ability to transform 3Y1 cells.

E6 (or E7) genes of some genital and cutaneous human papillomaviruses (HPVs) were compared for their ability to transform cells of a rat fibroblastic line (3Y1) by using recombinant retroviruses. The E6 gene of genital cancer-associated HPV 16 or 18 was found to induce a characteristic morphological change, i.e., densely packed arrays of elongated cells forming swirl patterns. This change is the same as that induced by the E6 gene of cutaneous cancer-associated HPV 5, 8, or 47, which we described previously. The E6 gene of HPV 1 or 11, associated with benign tumor of cutaneous or genital tissue, respectively, induced no such change. The E6 genes of all these cutaneous and genital HPVs enhanced anchorage-independent growth of the target cells induced by E7 gene of HPV 16 or 18, and this enhancing activity of HPVs 16, 18, and 47 was stronger than that of HPVs 1 and 11. A distinct type of morphological transformation of 3Y1 cells, i.e., rounded miniaturized cells that were densely packed without forming any distinctive arrays, was found to be induced strongly by E7 genes of HPVs 16 and 18, weakly by E7 of HPVs 1 and 11, and not at all by E7 of HPV 47. The results suggest that the intensity of the morphological change induced by E6 genes, rather than E7 genes, is correlated to the risk of malignant conversion of the lesion with which the corresponding HPVs are associated.