Tadao Funato

Alteration in Copy Numbers of Genes as a Mechanism for Acquired Drug Resistance

Chemoresistance is a major obstacle for successful treatment of cancer. To identify regions of the genome associated with acquired resistance to therapeutic drugs, we conducted molecular cytogenetic analyses of 23 cancer-cell lines, each resistant to either camptothecin, cisplatin, etoposide (VP-16), Adriamycin, or 1-β-d-arabinofuranosylcytosine, although the parental tumor lines were not. Subtractive comparative genomic hybridization studies revealed regions of gain or loss in DNA-copy numbers that were characteristic of drug-resistant cell lines; i.e., differences from their drug-sensitive parental cell lines. Thirteen ATP-binding cassette (ABC) transporter genes [ABCA3, ABCB1 (MDR1), ABCB6, ABCB8, ABCB10, ABCB11, ABCC1 (MRP1), ABCC4, ABCC9, ABCD3, ABCD4, ABCE1, and ABCF2] were amplified among 19 of the resistant cell lines examined. Three genes encoding antiapoptotic BCL-2 proteins (BCL2L2, MCL1, and BCL2L10) were also amplified and consequently overexpressed in three of the derivative lines. Down-regulation of BCL2L2 with an antisense oligonucleotide sensitized a VP-16 resistant ovarian-cancer cell line (SKOV3/VP) to VP-16. A decrease in copy numbers of genes encoding deoxycytidine kinase, DNA topoisomerase I, and DNA topoisomerase II α reduced their expression levels in one cytosine arabinoside-resistant line, two of three camptothecin-resistant lines, and two of five VP-16-resistant cell lines, respectively. Our results indicated that changes in DNA-copy numbers of the genes mentioned can activate or down-regulate them in drug-resistant cell lines, and that such genomic alterations might be implicated in acquired chemoresistance.

Rheumatoid arthritis after human parvovirus B19 infection

Polyarthropathy associated with human parvovirus B19 (B19) is usually transient, but often resembles rheumatoid arthritis (RA).1-3 However, B19 DNA screened by polymerase chain reaction (PCR) may be positive not only in autoimmune diseases, but also in non-arthropathy.4 To study whether or not RA occurs after acute B19 infection, we conducted a prospective study of patients with acute onset polyarthritis. Sixty seven cases showing acute inflammatory polyarthritis from January 1990 to June 1990 were examined and followed up for six years. Among them, 12 (9 female and 3 male) exhibited IgM anti-B19 antibodies.5 PCR, followed by a Southern blot analysis6 also showed the presence of B19 DNA in all 12 cases with IgM anti-B19 antibodies, but did not in the remainder. Initially, rheumatoid factor (RF) was negative in all B19 positive cases except one, but became positive in four, two to four months after the infection (table 1). Among RF …

Altered expression of retinoblastoma protein‐interacting zinc finger gene, RIZ, in human leukaemia

Summary. The retinoblastoma protein‐interacting zinc finger gene (RIZ), a member of the nuclear protein methyltransferase superfamily, is characterized by the presence of the N‐terminal PR domain. The RIZ gene encodes for two proteins, RIZ1 and RIZ2. While RIZ1 contains the PR (PRDI‐BF1 and RIZ homologous) domain, RIZ2 lacks it. RIZ gene expression is altered in a variety of human cancers and RIZ1 is now considered to be a candidate tumour suppressor. To investigate the role of RIZ in leukaemogenesis, we analysed the differential expression of RIZ1 and RIZ2 by quantitative real‐time reverse‐transcription polymerase chain reaction assay. Our results showed that the expression of RIZ1 was significantly decreased in leukaemia cell lines (14 out of 17, 82%) and in patients with acute myeloblastic leukaemia (eight out of 14, 57%). In contrast, RIZ2 expression was increased in patients with acute lymphoblastic leukaemia (eight out of 11, 73%), compared with normal bone marrow cells. These findings indicate that suppression of RIZ1 expression or enhancement of RIZ2 expression may have an important role in leukaemogenesis.

Complete XY gonadal dysgenesis and aspects of the SRY genotype and gonadal tumor formation

AbstractXY gonadal dysgenesis can be classified as either complete or incomplete according to gonadal morphology. The disease is a sex-reversal disorder resulting from embryonic testicular regression sequences and is induced by mutations in the sex-determining region Y (SRY) gene. The incidence of SRY mutations is thought to be approximately 20%. As the disease is characterized by a frequent complication of gonadal tumors, patients are usually advised to undergo prophylactic gonadectomy. In this study, we searched for mutations in SRY open reading frames from three patients with the complete form of XY gonadal dysgenesis, and detected missense mutations in two patients. Combined with the results of our previous study, in which SRY abnormalities were also detected in two out of three complete-type patients, the final incidence of SRY abnormalities was 67% (four of six patients), which is much higher than previously thought. The incidence of gonadal tumor formation in patients with SRY abnormalities was 50% (two of four patients), which is similar to the result of a metanalysis of patients with SRY abnormalities that revealed an incidence of 52.5%. Therefore, it is possible that the lower incidences of SRY abnormalities previously reported were caused by the inclusion of patients with the incomplete form or other sex-reversal disorders. Moreover, our results suggest that clinicians should carefully examine patients with SRY abnormalities.

Quantitative analysis of estrogen receptor alpha and beta messenger ribonucleic acid levels in normal endometrium and ovarian endometriotic cysts using a real-time reverse transcription-polymerase chain reaction assay

OBJECTIVE To quantify messenger RNA (mRNA) levels of the two estrogen receptor isoforms, estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) in the eutopic endometrium and ovarian endometriotic cysts. DESIGN Prospective study. SETTING University hospital. PATIENT(S) Patients with endometriosis and patients with uterine leiomyoma or carcinoma in situ. INTERVENTION(S) Gonadotropin-releasing hormone agonist (GnRH-a)-treated (n = 12) or untreated (n = 24) endometriotic cysts were obtained from 36 patients during laparoscopic cystectomy. Eutopic endometrium tissues were obtained from 24 patients during or immediately after surgery. MAIN OUTCOME MEASURE(S) ER-alpha and ER-beta mRNA levels, using a real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay, TaqMan RT-PCR. RESULT(S) Eutopic endometrium and ovarian endometriotic cysts showed predominantly higher levels of ER-alpha mRNA than ER-beta mRNA. Although ER-alpha and ER-beta mRNA levels in the eutopic endometrium were affected by a cyclic change in ovarian hormones, ovarian endometriotic cysts were less affected. Moreover, a long-term hypoestrogenic state induced by GnRH-a especially decreased ER-alpha mRNA levels in endometriotic cysts. Consequently, the relative ratios of ER-alpha to ER-beta mRNA levels in both GnRH-a-treated and untreated endometriotic cysts were significantly lower than those in the eutopic endometrium. CONCLUSION(S) The results suggest that the principal and regulatory effects of estrogens may be mediated mainly via ER-alpha rather than ER-beta in both the eutopic endometrium and endometriotic cysts.

Abnormal white matter lesions with sensorineural hearing loss caused by congenital cytomegalovirus infection: retrospective diagnosis by PCR using Guthrie cards

We report on two patients with congenital cytomegalovirus (CMV) infection asymptomatic at birth that was diagnosed retrospectively by polymerase chain reaction (PCR) of CMV DNA using blood stored on Guthrie cards. Neuroimaging studies showed abnormal myelination without any gray matter abnormalities. In the differential diagnosis of patients with abnormal white matter lesions and sensorineural hearing loss, one should consider congenital CMV infection. When investigating the etiology of patients with behavioral problems, migrational disorder, or white matter disease, PCR analysis of CMV DNA using blood stored on Guthrie cards might be helpful.

A quantitative reverse transcriptase polymerase chain reaction method for the detection of leukaemic cells with t(8;21) in peripheral blood.

Abstract: We evaluated the usefulness of a recently developed real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) system to detect minimal residual diseases (MRD) in patients with acute myelogenous leukaemia (AML) with chromosomal translocation t(8:21). The method was simple, rapid and reproducible for the quantity of chimeric AML1‐ETO (MTG8) transcripts. The ratio of the absolute copy number of a target gene (AML1‐ETO) to a control gene (glyceraldehyde‐3‐phosphate dehydrogenase, GAPDH) was calculated by using a fluorescence curve prepared from amplicons of serially diluted standard RNA. The relative points of MRD in bone marrow (BM) of 8 patients in the acute phase of the disease was from 0.85 to 3.0, whereas those of MRD in complete remission (CR) decreased to below 6.4×10−3. This method was also applied to evaluate chimeric transcripts in peripheral blood (PB) samples. The values in patients with t(8;21) AML were from 0.97 to 2.0 in the acute phase, whereas those in CR showed less than 2.2×10−4. There was 10−5‐fold difference in AML1‐ETO mRNA expression between PB samples in the acute phase and those in CR. The results suggest that we may easily monitor MRD in patients with t(8;21) AML through quantitative analysis of AML1‐ETO transcripts in blood samples.

Inhibition of motility and invasiveness of renal cell carcinoma induced by short interfering RNA transfection of β1,4GalNAc transferase

Human renal cell carcinoma (RCC) has been characterized by remarkable changes in ganglioside composition. TOS1 cells, typical of metastatic RCC, are characterized by predominance of GM2 as monosialoganglioside, and β1,4GalNAc disialyl‐Lc4 (RM2 antigen) as disialoganglioside [J. Biol. Chem. 276 (2001) 16695]. In order to observe the functional role of gangliosides in RCC malignancy, TOS1 cells were transfected with short interfering RNA (siRNA) based on open reading frame sequence of β1,4GalNAc transferase (β1,4GalNAc‐T), and its disordered sequence of siRNA (dsiRNA) as control. In siRNA transfectant, β1,4GalNAc‐T mRNA level and GM2 expression were greatly reduced, whereby GM3 expression appeared. In contrast, RM2 antigen level was unchanged, even though it has the same β1,4GalNAc epitope at the terminus. dsiRNA transfectant showed no change of β1,4GalNAc‐T mRNA and did not express GM3. Concomitant with reduction of GM2 and appearance of GM3, siRNA transfectant showed greatly reduced motility and invasiveness, although growth rate was unaltered. Both transfectants with siRNA and dsiRNA expressed the same level of tetraspanin CD9. Since CD9/GM3 complex is known to reduce integrin‐dependent motility and invasiveness [Biochemistry 40 (2001) 6414], it is plausible that motility and invasiveness of siRNA transfectant of TOS1 cells may be reduced by enhanced formation of such complex.

Increased sensitivity to cisplatin in gastric cancer by antisense inhibition of the her-2/neu (c-erbB-2) gene.

Background: The c-erbB-2 oncogene encodes a transmembrane tyrosine kinase receptor and its abnormal expression may be related to the prognosis of gastric cancer. Gastric cancer is relatively resistant to various drugs, including cisplatin. Cisplatin is widely used in cancer chemotherapy, but the mechanisms of drug resistance are not yet known. Methods: We used the human gastric cancer cell lines MKN-7 and KATO-III, which express the c-erbB-2 oncogene, as a model for relative resistance to cisplatin. We investigated whether inhibition with antisense oligonucleotides against c-erbB-2 increased the sensitivity of MKN-7 and KATO-III cells to cisplatin. Results: Antisense oligonucleotides for c-erbB-2 inhibited the expression of c-erbB-2 mRNA and protein and increased sensitivity to cisplatin, but not to other drugs, in MKN-7 and KATO-III cells. Cell growth was also inhibited by c-erbB-2 antisense oligonucleotides but not sense oligonucleotides. Conclusions: These findings indicate that c-erbB-2 expression in gastric cancer is one of the factors related to cisplatin sensitivity, and that anti-c-erbB-2 antisense oligonucleotides induced increased sensitivity to cisplatin.

In vitro leukemia cell models of Ara-C resistance

Cells of the human leukemia line K562 were continuously exposed to cytosine arabinoside (Ara-C) at increasing concentrations for 3 months. The resulting cell line, termed K562/AC, showed 48-fold resistance to Ara-C, compared with the parental K562 cells. The sensitivities of K562/AC to adriamycin (ADR), vincristine (VCR) and etoposide (VP16) were similar to those of parental K562. Gene analysis revealed that this cell line lacked expression of the deoxycytidine kinase (dCK) gene, which was evident in Ara-C-sensitive cells. As in K562 cells, multidrug resistance (MDR-1) and multidrug resistance protein (MRP) genes were not expressed in K562/AC. We also established an in vitro model of Ara-C resistance using phosphorothioate antisense oligonucleotides to dCK (dCK-AS). Treatment of K562 with dCK-AS caused decreased dCK expression and 6- to 10-fold increases in resistance to Ara-C, compared with that in cells treated with sense oligonucleotides to dCK (dCK-S) or in non-transfected cells. The cells described here may contribute to the study of a novel mechanism associated with Ara-C resistance, in which reduced dCK activity may play an important role.