Development of highly nutritive culture media.

A highly nutritive culture medium (MGM-464) was developed for insect cell primary culture. The new medium consists of 6 inorganic salts, 4 organic acids, 21 amino acids, 3 sugars, 10 vitamins, and 8 other chemicals, including natural substances. The complete medium was generated by adding 20 ml fetal bovine serum to 100 ml MGM-464. The detail of the composition of the medium is given in a table, and the protocol to prepare the medium is described in the text. Among the 15 kinds of cultures made with MGM-464, embryonic cells from a walking stick and ovarian cells from the common white were subcultured more than 70 times, and embryonic cells of a chrysomelid beetle were subcultured more than 15 times. Other cultures could not be subcultured. However, embryonic cells from the commercial silkworm and a cockroach, ovarial cells from the commercial silkworm and a sphingid moth, nervous cells from the commercial silkworm and two sphingid moths, and cells from the dorsal vessel plus surrounding tissue of the commercial silkworm survived for several mo. The cells from the honeybee embryos, aphid embryos, and planthopper embryos were rather short-lived, and deteriorated after about 1 mo.

Vitamin requirements of the cultured flesh fly cells, Sarcophaga peregrina (Diptera, Sarcophagidae).

Essential vitamins for the growth of a cell line derived from the flesh fly, Sarcophaga peregrina, were determined. By examining the survivability of continuous passages of the cells in the chemically defined medium lacking one vitamin, thiamine, riboflavin, pantothenate and either niacin or niacinamide were found to be essential for the continuous growth of the flesh fly cells in vitro. [Originally published in Volume 37, Archives of Insect Biochemistry and Physiology, 37:283-286 (1998).]

Sperm reflux and its role in multiple mating in males of a butterfly Polygonia c-aureum Linnaeus (Lepidoptera: Nymphalidae).

The relationship between sperm quantity in the duplex and that in the vasa deferentia was examined in the Asian comma butterfly, Polygonia c-aureum. In virgin males, the number of eupyrene sperm bundles in the duplex increased linearly with age, whereas that in the vasa deferentia was consistently small. However, numerous sperm were found in the vasa deferentia of males immediately after mating. The number of eupyrene sperm bundles in the vasa deferentia after mating significantly increased with age and with increasing the time interval between matings. From these and other results, it was suggested that some sperm in the duplex were moved back to the vasa deferentia during mating, and that such sperm reflux provides a means to save sperm for multiple mating.

Effect of culture medium on the in vitro secretion activity of prothoracic glands from Pseudaletia separata.

The prothoracic glands (PGs) taken from the last instar of the common armyworm, Pseudaletia separata, were cultured in various media for the purpose of finding a suitable medium for relatively long-term culture of PGs. Among the tested culture media, MGM-450 medium without serum was the best to maintain PG cells viable for relatively long periods, and to continue to secrete ecdysteroids. Secretion of ecdysteroid by the PG in vitro became marked when the PG was taken from last instar larvae older than 2 days after the last molt. PGs cultured in any of the media secreted ecdysteroid only within the first 2 h after placing them in culture, however, in the MGM-450 medium, the PGs secreted ecdysteroid even after 5 days of culture.

Mechanisms for 5-fluorouracil resistance in human colon cancer DLD-1 cells.

A 5-fluorouracil (5-FU)-resistant subline, DLD-1/5-FU, was established by repeated 5-d exposures of human colon cancer DLD-1 cells to 5-FU. DLD-1/5-FU cells were 41- and more than 75-fold resistant to 96-h and 1-h exposures to 5-FU, respectively. When exposed to 5-FU, DLD-1/5-FU cells exhibited marked resistance to in situ thymidylate synthase (TS) inhibition by 5-FU as compared to DLD-1 cells, and incorporation of 5-FU into cellular RNA in DLD-1/5-FU cells decreased to 25% of that in DLD-1 cells. As causes of resistance to DNA and RNA-directed actions of 5-FU, remarkable reduction of intracellular levels of both 5-fluorouridine 5'-triphosphate (FUTP) and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) in DLD-1/5-FU cells was confirmed. It was found that activities of uridine kinase, orotate phosphoribosyltransferase and thymidine kinase of DLD-1/5-FU cells were significantly lower than those of the parent cells. Intracellular levels of TS were similar between the two cell lines. These results indicated that the mechanism of resistance to 5-FU in DLD-1/5-FU cells involves reduced enzymatic activation of 5-FU.

Effects of Environmental Conditions and Aging on Eupyrene Sperm Movement in Male Adults of Polygonia c-aureum (Lepidoptera: Nymphalidae).

The effects of temperature, photoperiod and aging on eupyrene sperm movement from the testis to the duplex in Polygonia c-aureum male adults were examined in relation to seasonal form and imaginal diapause. In males of both summer and autumn forms obtained under long-day and short-day conditions, respectively, the number of eupyrene sperm bundles in the duplex increased linearly with age during the early stage of adult life at 21 degrees C, and no marked difference was observed between the two seasonal forms. Photoperiod during the adult stage did not influence the rate of sperm movement in autumn forms with diapause. The lower the temperature, the smaller the number of sperm bundles moved from the testis to the duplex. Sperm movement did not appear to occur during a period of chilling at 5 degrees C. Males of the autumn form which were pre-incubated for 30 days and chilled for 60 days failed to resume rapid sperm movement at the final incubation temperature of 21 degrees C. Those which were pre-incubated only for 15 days at 21 degrees C had much fewer sperm bundles in the duplex after chilling than those pre-incubated for 45 days. These results suggest the possibilities that eupyrene sperm movement is suppressed strongly during and after overwintering in the field, and that the eupyrene sperm to be used for reproduction in spring are transferred from the testis to the duplex before overwintering.

An insect cell line discrimination method by RAPD-PCR.

RAPD-PCR with a tenmar single primer for discrimination of insect cell lines was devised. The base sequence of the primers used were TTCGAGCCAG, CGGCATCTAC, GAACGGACTC, and TGAGTGGGTG (GC contents were 60%). Genome DNA was extracted by modified Landry et al. (1993) method. The reaction mixture consisted of 10 microliters buffer, 8 microliters dNTP mixture (2.5 mM each), 4 microliters primer (50 microM), Taq DNA polymerase (2.5 units), 1 microliter template DNA; and the reaction was run at 94 degrees C for 2 min (denaturation), followed by 31 cycles of 94 degrees C for 1 min, 42 degrees C for 1 min (annealing), and 72 degrees C for 2 min (extension) and terminated with 72 degrees C for 7 min. By developing the reaction products with agarose gel electrophoresis, it became evident that DNA fragments were amplified with all the primers used. Among four primers, the second primer was selected as a suitable primer for distinguishing cell lines. With this method, cell lines derived from different species were clearly distinguished.

Production of rotenone-inactivating substance(s) by rotenone-resistant insect cell line.

In a previous paper, we showed that a cell line derived from hemocytes of the cabbage armyworm, Mamestra brassicae (R-cell) was a thousand times as resistant to rotenone as that from ovaries of the same species (S-cell). The S-cells were killed by rotenone at concentrations higher than 10(-9) M, while R-cells at higher than 10(-6) M. When the R-cells were cultured in the medium containing 10(-9) M rotenone, the ability of rotenone to kill the S-cells was lost in the used medium. Also, when rotenone was incubated in the medium conditioned with R-cells, it lost its cell killing activity. It became evident that rotenone-inactivating substance(s) were produced in cells and stored in water-soluble form or liberated into the medium. The substance(s) were inactivated by heat treatment.

Reduced activity of anabolizing enzymes in 5-fluorouracil-resistant human stomach cancer cells.

The mechanism of resistance to 5-fluorourcil (5-FU) was studied with NUGC-3/5FU/L, a human stomach cancer cell line which had acquired resistance as a consequence of repeated 5-day exposures to stepwise-increasing concentrations of 5-FU in vitro. NUGC-3/5FU/L was 200-fold and over 16-fold resistant to 96-h and 1-h exposures to 5-FU, respectively. NUGC-3/5FU/L incorporated less 5-FU into RNA, indicating resistance to the RNA-directed action of 5-FU. On the other hand, NUGC-3/5/5FU/L also showed resistance to in situ thymidylate synthase (TS) inhibition by 5-FU. Polymerase chain reaction-single-strand conformation polymorphism analysis of TS cDNA and a FdUMP ligand binding assay showed that quantitative and qualitative alterations of TS are not responsible for this resistance. In contrast, the ability to metabolize 5-FU to its active metabolites, FUTP and FdUMP, was reduced in NUGC-3/5FU/L. We found that not only the activities of uridine phosphorylase/kinase and orotate phosphoribosyl-transferase (OPRT), but also the level of phosphoribosyl pyrophosphate, a cosubstrate for OPRT, were significantly lower in NUGC-3/5FU/L than in the parent NUGC-3. These results indicated that resistance to 5-FU in NUGC-3/5FU/L is due to reduced activities of 5-FU-anabolizing enzymes, but not to an alteration of TS. 2'-Deoxyinosine effectively enhanced TS inhibition by 5-FU in the resistant cells, thus markedly sensitizing them to 5-FU.

Production of ethanol by cultured insect cells.

Proton Nuclear Magnetic Resonance (1H-NMR) Analysis of insect cell culture media used for cultivating insect cell lines derived from the fleshfly Sarcophaga peregrina, swallowtail butterfly Papilio xuthus, and cabbage armyworm Mamestra brassicae revealed that ethanol appeared in the medium as the cultures aged. By incorporating [13C-1]-glucose into the media, we pursued 13C-NMR spectrograms to show that the ethanol was derived from glucose. Thus, it became evident that the insect cells cultured in vitro produce ethanol from glucose as a metabolite.

Different modes of cell-killing action between DNA topoisomerase I and II inhibitors revealed by kinetic analysis.

We compared the modes of cell-killing by DNA topoisomerase I and II inhibitors. The effects of camptothecin (CPT), KT-6528 and UCE6 upon colony formation by inhibiting DNA topoisomerase I, and of etoposide (VP-16), teniposide, amsacrine and UCT4-A as inhibitors of DNA topoisomerase II were analyzed based upon a kinetic method that distinguishes between cell cycle phase-specific and -nonspecific agents. Human colorectal cancer WiDr cells were exposed to several concentrations of each agent for various periods and 90%-inhibitory concentrations (IC90) at each time were determined by means of a clonogenic assay. When exposure times and corresponding IC90s were plotted on a log-log scale, all inhibitors of DNA topoisomerase II gave curves including a linear portion with a slope of -1, which is characteristic of cell cycle phase-nonspecific agents. In contrast, the curves for all inhibitors of DNA topoisomerase I had a much steeper slope than -1, which is typical of cell cycle phase-specific agents. In agreement with this finding, the cells were remarkably accumulated in the G2-M phase when exposed to VP-16, but in late S-phase when exposed to CPT as determined by a flow cytometric assay. These results indicated that the two classes of agents kill cells in a quite different manner although they are inhibitors of similar enzymes.

Flow cytometric analysis of cell-killing actions of 5-fluorouracil in human colorectal cancer cells.

To confirm our previous kinetic analysis of the mode of cell-killing action of 5-fluorouracil (5-FU), we carried out a flow cytometric analysis with human colorectal cancer DLD-1 cells. Cells were treated with each cytotoxic concentration of 5-FU for 1 or 72 h, and the periodic changes in flow cytometric pattern were compared with those of 5-fluorouridine (FUrd) and 5-fluoro-2'-deoxyuridine (FdUrd). When cells were cultured with 5-FU for 72 h, most of them accumulated in S phase and remained there. This pattern was the same as that seen in cells that were continuously exposed to FdUrd. In contrast, when cells were exposed to 5-FU for 1 h and cultured in drug-free medium, they ended the cell-cycle traverse in either G2/M or G1 phase after an immediate but transient accumulation in S phase. Results were identical to those observed with cells similarly treated with FUrd. These results demonstrated a good accordance with those of our kinetic analysis, and strongly suggested that the mode of cell-killing exhibited by 5-FU differs with exposure time: 5-FU acts like FUrd in short exposure conditions, and it acts similarly to FdUrd in continuous exposure conditions.

Therapeutic efficacy of combination of antitumor agent with AHC-52 against multidrug-resistant cells in the intravenously inoculated P388 leukemia model.

To predict the clinical effect on leukemic disease of a combination regimen developed to circumvent multidrug resistance (MDR), we tested various antitumor agents in the presence and absence of AHC-52, a sensitizing agent for multidrug-resistant cells, in the i.v.-i.v. model of murine leukemia. In this model system, sensitive and resistant P388 murine leukemia cells are inoculated i.v. into mice, and each antitumor agent is injected via the i.v. route. Vincristine (VCR) had no effect on the survival of mice bearing VCR-resistant P388, a relatively poorly resistant subline, when given either as a single agent or in combination with AHC-52. In contrast, adriamycin (ADR) alone had no effect on these mice, but its combination with AHC-52 resulted in significant survival, the maximal value achieved being 196% (treated mice/control animals, T/C). Etoposide (VP-16) strongly enhanced survival, even when used alone, and this effect was markedly potentiated by AHC-52. Combination of any antitumor drug with AHC-52 was ineffective in mice bearing ADR-resistant P388, a highly resistant subline. On the other hand, AHC-52 strongly augmented the therapeutic efficacy of these antitumor agents in mice bearing the sensitive parent P388 leukemia, producing some curative effects. On the basis of these results, the feasibility of this type of combination therapy is discussed.

Mechanism for resistance to 5-fluorouracil in P388 leukemia cells.

In order to assess mechanisms for acquired resistance to 5-fluorouracil (5-FU) of P388 cells on a cellular basis, we compared sensitivities of P388 and its 5-FU-resistant subline (P388/5-FU) cells to 5-FU, 5-fluorouridine (FUrd) and 5-fluoro-2'-deoxyuridine (FdUrd). P388/5-FU cells exhibited an approximately 10-fold resistance to 5-FU and 170-fold cross-resistance to FUrd but not to FdUrd when they were exposed to each agent for 5 h in vitro. 5-FU-induced growth inhibition was hardly reversed with thymidine, suggesting its ribonucleic acid (RNA)-directed effect. This was supported by the fact that similar amounts of 5-FU were incorporated into cellular RNA in P388 and P388/5-FU when these cells were incubated with equitoxic concentrations of 5-FU. Furthermore, incorporation of 5-FU and FUrd into cellular RNA in P388/5-FU cells were significantly lower than in P388 cells when cells were exposed to them at the same concentration. These results suggest a major action of 5-FU is directed toward RNA in these cells at least under the present experimental condition, and 5-FU resistance of this cell line is closely associated with reduced uridine kinase activity among various enzymatic changes previously observed.

Kinetic analysis of 5-fluorouracil action against various cancer cells.

Based on our recent kinetic analysis, which made it possible to distinguish between the cell-killing actions of cell cycle phase-specific and non-specific agents, we attempted to elucidate the actions of 5-fluorouracil (FUra) on three different cancer cell lines. By colony-forming assay, the concentrations of fluorouridine (FUrd), fluorodeoxyuridine (FdUrd) or FUra giving 90% cell kill (IC90) at various exposure times (texps) were obtained. With P388 cells, the curve of texps-IC90 for FUrd on a log-log scale was linear with a slope of -1, which is typical for cell cycle phase-nonspecific agents. In contrast, the curve for FdUrd showed a much steeper slope than -1, which is characteristic for cell cycle phase-specific agents. We found that the curve for FUra was exactly the same as that for FUrd, indicating that the mode of FUra action on P388 leukemia is analogous to that of FUrd. Similar results were observed with human colon and renal cancer cell lines, HT-29 and KU-2, although when the cells were exposed to relatively low concentrations of FUra for a long time, a cell cycle phase-specific action became evident.

A benzophenazine derivative, N-beta-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxy-benzo[a]phenazine-6-carboxamide, as a new antitumor agent against multidrug-resistant and sensitive tumors.

NC-190, a benzophenazine derivative (N-beta-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxy-benzo[a]phenazine-6-carboxamide), was effective against multidrug-resistant human and mouse tumor cells in vitro and in vivo. When vincristine (VCR)-resistant P388 leukemia-bearing mice were treated with an optimal dose of NC-190, four of six mice were cured, whereas treatment of mice with VCR resulted in only a marginal increase in life span. The compound also showed chemotherapeutic effect against Adriamycin-resistant P388 leukemia-bearing mice and was effective against various multidrug-resistant human and murine tumor cells in vitro. Its cytotoxicity to multidrug-resistant K562 cells was not enhanced by the addition of verapamil. The accumulation of NC-190 in multidrug-resistant K562 cells was slightly lower than that observed in sensitive K562 cells; the compound did not efficiently inhibit the binding of VCR to the plasma membrane of resistant cells, indicating that NC-190 has little affinity for P-glycoprotein. NC-190 inhibited the activity of DNA topoisomerase II. These observations suggest that NC-190 (1) is not transported out of resistant cells by P-glycoprotein and (2) inhibits DNA topoisomerase II activity in the cells, resulting in its likely effectiveness against various multidrug-resistant tumor cells.