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Background: CHIP is a U box type ubiquitin ligase that induces ubiquitination and degradation of its target proteins, which include several oncogenic proteins. We clarified that CHIP suppresses tumor growth and metastasis in human breast cancer by inhibiting oncogenic pathways (Nat Cell Biol 2009;11:312-9). In this study, we investigated the relationship between immunohistochemical CHIP expression and several biomarkers, and evaluated the correlation between CHIP expression and prognosis of patients with invasive breast cancer. Patients and Methods: In this study, 272 consecutive patients with invasive carcinoma of no special type with invasion >5 mm, treated in 2000-2001, were enrolled. We performed immunohistochemical staining with antibodies against CHIP, ER, PgR, and HER2. Cytoplasmic expression patterns of CHIP were scored as 0 (no stain), 1 (weak stain), 2 (moderate stain), and 3 (strong stain), and divided into high CHIP (score 3) and low CHIP (score 2, 1, 0) groups. We investigated the relationship between CHIP expression and ER, PgR, HER2, histological grade (HG) and lymph node status (pN). We also evaluated correlation between CHIP expression and prognosis (RFS) of patients. Results: CHIP expression was as follows: score 0, 49 patients (18.0%); score 1, 91 (33.5%); score 2, 83 (30.5%); score 3, 49 (18.0%). High CHIP correlated significantly with positive ER (p Conclusions: CHIP expression correlated with status of hormone receptors and HER2 and might be a potent prognostic factor in invasive breast cancer patients, especially postmenopausal cases. Immunohistochemical examination of CHIP is thought to be useful for evaluating prognosis of breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-18.

[Background] Estrogen receptor (ER) positive breast cancer was composed of about 70% of clinical breast cancer and therapy for the ER positive breast cancer have progressed, but some patients recur after endocrine therapy including aromatase inhibitors (AIs). The switch of proliferation drive to human epidermal growth factor receptor 2 was reported as one of AI resistance mechanisms, but ER status of many recurrent patients after AI treatment was maintained or slightly decreased. Moreover, plasma concentration of estrone-sulfate (E1S) is mostly unchanged after menopause and treatment of AI. Thus, we established AI resistant model with conserved ER expression and its activity from MCF-7 cell, and found that E1S contributed to proliferation of the AI-resistant cells. [Material and Method] We introduced plasmids carrying ERE-green fluorescent protein (GFP) gene to MCF-7 (E10), and then aromatase gene was additionally introduced to E10 (E10arom). E10arom was cultured in estrogen-depleted medium including testosterone and letrozole, monitoring GFP as ER activity for about three months. We finally established ER positive Letrozole-Resistant cell lines (LR cells). In addition, to confirm whether E1S-dependent growth in LR cells could be reproduced in ER positive breast cancer cell line, we also established human steroid sulfatase (STS) gene-transfected MCF-7 (MCF-STS) and organic anion transporter peptide (OATP) 4A1 gene-transfected MCF-7 (MCF-4A1). ER activity of these cell lines was examined by ERE-luciferase reporter assay, and response to each steroid and drug was investigated by cell proliferation assay. Quantitative PCR was used for comparing expression of mRNA in these cell lines. [Results] mRNA expression of STS and four OATPs including OATP4A1 increased in LR cells comparing to E10arom. Moreover, there was no change in mRNA expressions of efflux drug transporters, aromatase-metaboic enzyme and enzymes related with production for estrogenic androgen. ERE-luciferase assay with E1S showed higher ER activity of LR cells than E10arom, and LR cells were more proliferated by E1S in dose-dependent manner than the parental cell. STS inhibitor alone and its combination with letrozole were inhibitory to proliferation of LR cells. LR cells were also inhibited by selective estrogen receptor modulator (SERM) and downregulator (SERD). In proliferation assay of MCF-STS and MCF-4A1, we observed that these cell lines also proliferated by E1S in dose-dependent manner as LR cells did. [Discussion] Since ER status of recurrent patients after AI treatment was mostly unchanged, our model could be more practical for analysis of AI resistance in ER positive breast cancer. These results showed that combined induction of STS and OATP contributed to estradiol (E2) production from E1S in LR cells, suggesting that analysis of E1S as alternative E2 productive pathway needed investigation of both STS and transporter of E1S. The combination of STS inhibitor with letrozole was effective to LR cells, and SERM and SERD were also effective to these cells, suggesting that combined therapy of STS inhibitor with AI or switching AI to SERM or SERD was beneficial for AI-resistant breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-16.

Aromatase inhibitors (AI) are the common treatment for postmenopausal estrogen receptor (ER) positive breast cancer. However, not a few of these patients acquire resistance to AI.To investigate this molecular mechanism of resistance, we have established several breast cancer cell lines as AI resistant models. Recently, we reported on AD-EDR cells (androgen metabolite-dependent and estrogen depletion resistant cells) derived from MCF-7, in which ER activity depends on one of androgen metabolite, 5a-androstane-3β, 17β-diol (3βdiol). It is supposed that tumor heterogeneity contributes to the acquired resistance to drugs.To examine this diversity, we established another AI resistant model derived from T-47D, to compare characters of these AI resistant models from different parents. Then, we found that the androgen receptor (AR) signal acquired oncogenic role in this model in contrast to AD-EDR cells established from MCF-7. We cultured ER positive breast cancer cells, T47D-TE8, stably transfected with ERE-GFP reporter into T47D, under estrogen-depleted and androgen-supplemented conditions for more than three months, simulating the AI treatment, and established estrogen deprivation-resistant cells (TE8-EDR cells). TE8-EDR cells had little ERE-GFP activity and proliferation response to estrogen though estrogen stimulated proliferation of parental T47D-TE8. The mRNA for ER and progesterone receptor was hardly detected in TE8-EDR cells. These results suggested that they had lost the ER-mediated pathway of growth. On the contrary, Dihydrotestosterone (DHT) increased growth of T47D-TE8 cells, while it was more remarkable in TE8-EDR, and this proliferative effect was reduced by AR antagonist, bicalutamide. The mRNA expressions of AR and its target gene, PSA, were extremely increased in TE8-EDR cells. These results indicated that TE8-EDR enhanced AR-regulated pathway for growth. Increased AR expression was presumed to be one of the factors elevating AR activity. To identify other elements enhancing AR-mediated signal, we compared the gene expression of TE8-EDR cells with that of T47D-TE8 by microarray analysis. The outcome showed that the expression of some well-known androgen-related genes were increased in TE8-EDR cells. Among these genes, we focused on DDC (L-dopa decarboxylase) which has been reported as one of the AR coactivator, and its overexpression increase proliferative effect in prostate cancer cell lines.By real-time RT-PCR, we confirmed that the mRNA expression of DDC in TE8-EDR was higher over one-hundred fold than T47D-TE8. In addition, DHT-induced proliferation was cancelled by DDC inhibitor, NSD-1015. This study suggests that TE8-EDR cells shift their growth pathway from ER to AR signal, and the AR activity is enhanced by high-expression of DDC. Whereas, MCF7-derived AD-EDR cells still depends on ER signal for their proliferation. So far, few studies have shown the proliferative effects of androgen as AI resistant mechanism. Our results provide new mechanism of acquired AI-resistance in breast cancer. Moreover, the fact that the character of AI resistant models varied with their parent cells supports the hypothesis that tumor heterogeneity contributes to diversify the mechanism of acquired resistance to hormonal therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-10.

Background: CDK4/6 inhibitors have received FDA breakthrough therapy designation as 1st line treatment for advanced estrogen receptor positive breast cancer patients. However, the benefit offered by CDK4/6 inhibitors is individually different and furthermore acquired resistance to the drugs is monumental challenges. It is urgent need to search for the biomarker and understand the drug sensitivity and its alteration after acquired resistance. Results: To identify the efficacy of CDK4/6 inhibitors, we assessed IC50 of ribociclib in several cell lines. Luminal cell lines (MCF-7, T-47D) exhibited lower ribociclib IC50 than HER-2 (SK-BR-3) and triple negative cell lines (MDA-MB-231, BT-20). Immunoblot analysis of Luminal cell lines showed extremely lower levels of CDK6 compared with others. CDK6-transfected MCF-7 by means of expression vector reduced the sensitivity equivalent to MDA-MB-231 not only to ribociclib but also to palbociclib and abemaciclib. Protein level of ERα in CDK6-transfected MCF-7 stayed unchanged and fulvestrant sensitivity was unaltered as well. Subsequently, we detect the efficacy of ribociclib in hormone resistant cell lines. Estrogen deprivation-resistant (EDR) cells (EDR1:ER-positive, EDR2:ER-negative) and fulvestrant resistance (MFR) cells (loss of ER expression) established from MCF-7 maintained ribocilib sensitivity to the same degree with MCF-7. No marked difference in IC50 was observed between EDR1/2 and MFR, and CDK6 expressions were comparable to MCF-7. These results suggest that high level of CDK6 expression weaken the sensitivity to CDK4/6 inhibitors. The inhibitors would provide more effective benefits to tumors expressing lower level of CDK6 than the higher, independent of hormone sensitivity. To understand the characteristics in acquired resistance, ribociclib resistant cell lines (RIBR1/2) were established from EDR1 by long-term culture with ribociclib. RIBR designed lower level of p21, p27 and ERα by immunoblot analysis. EDR1 were promoted cell growth by estrogen addition, while RIBR were not. Further, ER activity of RIBR was intensely decreased, and mRNA levels of the ER target genes, PgR and EGR3 were also decreased. Therefore, the responsiveness to tamoxifen and fulvestrant were lost. On the other hand, PI3K inhibitor and mTOR inhibitor suppressed cell growth to the same extent as EDR1, suggesting that RIBR were reduced ER dependence and remain reliant on PI3K/Akt/mTOR pathway. Conclusion: The possibility of CDK6 as a biomarker is corroborated by the finding that low level of CDK6 expression is positively correlated with the efficacy of CDK4/6 inhibitor. Further ER dependence had decreased after acquired CDK4/6 inhibitor resistance whereas the dependence on PI3K/Akt/mTOR pathway still remain, indicating the inhibition of PI3K/Akt/mTOR pathway would be amenable to therapeutic target. Citation Format: Iida M, Nakamura M, Tokuda E, Niwa T, Ishida T, Hayashi S-I. CDK6 might be a key factor for efficacy of CDK4/6 inhibitor and the hormone sensitivity following acquired resistance [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-04-02.

Background Mutations in phosphatidylinositol 3-kinase (PI3K), which encodes the catalytic subunit of PI3Kα, is one of the most frequent genomic alterations and is found in about 40% of estrogen receptor (ER)-positive, HER2 negative breast cancers. PIK3CA mutations promote the growth and proliferation of cancers via activation of the PI3K/mammalian target of rapamycin pathway and can mediate resistance to endocrine therapies in breast cancer. Although several clinical trials for PI3K inhibitors (PI3Kis) in ER-positive metastatic breast cancers are ongoing, the effects of PI3Kis on endocrine-resistant breast cancers with various characteristics and definitive biomarkers of PI3Kis are unclear. Using endocrine-resistant cells established in our laboratory, we evaluated the efficacy of PI3Ki in these cell lines and identified the characteristics associated with acquired resistance to PI3Kis in endocrine-resistant cells. Results Long-term estrogen deprivation-resistant (EDR) cell lines and fulvestrant-resistant cell lines (MFR and TFR) were established from MCF-7 and T-47D cells in our previous studies. These cell lines showed different ER expression levels, including high expression (EDR-1), low expression (EDR-2, -3), or no expression (MFR, TFR); all of these cell lines had the same PIK3CA mutations as the parental cell lines. The pan-class1 PI3Ki buparlisib (BKM120) and α-specific PI3K inhibitor alpelisib (BYL719) inhibited the proliferation of endocrine-resistant cell lines when compared with their parental cells. Among endocrine-resistant cells, MFR cells were dramatically inhibited by PI3Kis. Colony formation assays indicated that MFR cells were more sensitive to PI3Kis than other cells lines. Next, we established PI3Ki- and everolimus-resistant cell lines from EDR-1, EDR-2, and MFR cells using BKM120, BYL719, and everolimus. Analysis of the time required to generate resistant cells showed that MFR cells required twice as long to acquire resistance compared with EDR cells. Furthermore, the time required to acquire resistance to BYL719 was shorter than that for BKM120. BYL719-resistant (BYL-R) cells were effectively inhibited by BKM120 to a degree similar to that of parental cells; however, BYL-R cells lost sensitivity to BYL719 and everolimus. Evelolimus-resistant (EVE-R) cells were also the same. In contrast, BKM120-resistant (BKM-R) cells showed less sensitivity to BKM120, BYL719, and everolimus. In other words, the pan-PI3Ki BKM120 was able to inhibit the growth of BYL-R and EVE-R cells, whereas BYL719 and everolimus were not able to inhibit BKM-R cells sufficiently. In addition, there were no changes in ER expression in EDR-1, EDR-2, and MFR cells exposed to PI3Kis for 1 h. Interestingly, ER expression on EDR-2 cells with acquired resistance to PI3Kis was increased compared with that in parental cells. Conclusion Our findings showed that PI3Kis exhibited remarkable efficacy in all types of EDR cells, particularly in fulvestrant-resistant cell lines. In PI3Ki- and everolimus-resistant cell lines, BKM120 and BYL719 showed different effects, and BYL719 and everolimus may exhibit cross-resistance. Furthermore, PI3Kis were likely to change the expression of ER. Citation Format: Tokuda E, Komatsu T, Sasaki S, Tsuboi K, Nakamura M, Iida M, Niwa T, Saito M, Hayashi S-I. Effects of PI3K inhibitors on endocrine-resistant cell lines and differences in the characteristics of ER positive breast cancer cells after acquired resistance to the inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-12.

To investigate molecular mechanisms of growth control by diets, we examined the effects of nutrition on the expression of c-myc and insulin-like growth factor-1 (IGF-1) genes in the liver of growing rats. In the first study, rats were fed either a 24% casein, 24% zein or protein-free diet, or were starved for 3 d. The levels of the two mRNAs in the tissues were then determined by Northern blot hybridization. In the liver, levels of the two mRNAs varied in a reciprocal anner in response to changes in either quantity or quality of diet. The expression of c-myc mRNA was greatly enhanced by consumption of the protein-free diet or by starvation, whereas the IGF-1 mRNA levels were reduced markedly by consumption of the zein diet or the protein-free diet or by starvation. In another study, the casein and zein diets were fed to rats that had been adapted to a 2-h meal-feeding pattern, first with nonpurifled diet for 10 d and then with the protein-free diet for 3 d before the experiment. In rats fed casein, the level of c-myc mRNA decreased 75% within 8 h after consumption of the casein diet, whereas the IGF-1 mRNA level increased 100% during that period. Consumption of the zein diet did not affect the level of either mRNA. Because quantity of food intake did not differ between the rats fed casein and those fed zein, expression of the two genes in the liver was affected by the quality of the protein consumed. These results indicate that quality and quantity of diets changed the expression of c-myc and IGF-1 genes and thus demonstrate the possibility that nutrition not only supplies material for body components but also affects the signal transduction for growth in young growing rats.

The effect of the 5'-untranslated region of ornithine decarboxylase (ODC) mRNA and of spermidine on the translation of ODC mRNA was studied in a rabbit reticulocyte cell-free system. The ODC mRNAs, possessing different sizes of the 5'-untranslated region and named mODC241, mODC188, mODC85, and mODC24 according to the number of nucleotides in the 5'-untranslated region, were synthesized by in vitro transcription techniques through the use of plasmids containing various sizes of mouse ODC cDNA. As the size of the 5'-untranslated region of ODC mRNA increased, the efficiency of the translation decreased and the degree of stimulation of the translation by 0.2 mM spermidine increased. The inhibition of ODC mRNA translation by high concentrations of spermidine (0.6-1 mM) also increased with an increase in the size of the 5'-untranslated region. The spermidine effects were increased greatly when the size of the 5'-untranslated region of ODC mRNA was increased from 85 to 188 nucleotides. These results suggest that the nucleotides 70-220 upstream from the initiator AUG are involved in the decrease in translational efficiency. In addition, at least the nucleotides in the 70-170 region upstream from the initiator AUG are important in the strong stimulation of ODC synthesis by low spermidine concentrations and in the inhibition of ODC synthesis at high spermidine concentrations.

A DAC with a 500MHz conversion rate, 2ns settling time, 0.6ns rise/fall times and 200ps/V glitch energy will be reported. The DAC has been fabricated in shallow groove VLSI technology.

One of the tools to test an in vitro hypothesis in vivo is transgenic/gene targeting technology. Transgenic technology provides many advantages such as: (1) the study of specific gene function as systemic and developmental effects; and (2) testing of specific gene function chronically. nevertheless, one disadvantage of this technology is the difficulty in tissue-specific targeting of the transgenic expression. Another useful tool is the in vivo gene transfer approach. Therefore, we tested a potential novel model for the study of transgene expression and knock-out using antisense technology. Here, we have demonstrated that incubation of hemagglutinating virus of Japan (HVJ)-liposome complex containing FITC-labeled oligonucleotides in the amniotic fluid of fetal rats resulted in the nuclear localization of fluorescence in the skin (epidermis and dermis). Similarly, transfection of beta-galactosidase gene resulted in positive staining in several surface layers of the skin. Thus, local gene or antisense oligonucleotide transfer approach into the skin may be useful for studying the role of autocrine/paracrine mediators and treating diseases.

Introduction: Aromatase inhibitors (AIs) have been reported to exert their anti-proliferative effects not only by reducing the production of estrogens but also by unmasking the inhibitory effect of androgens such as testosterone (TS) or dihydrotestosterone (DHT) in postmenopausal women with hormone receptor-positive breast cancer. The behavior of androgens in AI-resistance mechanisms is not sufficiently understood. 5α-androstane-3β,17β-diol (3β-diol) generated from DHT by 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD type 1: HSD3B1) has androgenic activity and substantial estrogenic activity, representing a potential mechanism of AI resistance. Methods: To investigate these issues, ERE-GFP-transfected MCF-7-E10 cells were cultured for 3 months under steroid-depleted, TS-supplemented conditions which is the similar as the AI treatment. Among the surviving cells, two stable variants that show ER activity depending on androgen metabolites were selected as AD-EDR (androgen metabolite-dependent and estrogen depletion-resistant) by monitoring GFP expression. Using these cell lines, we investigated the process of adaptation to androgen-abundant conditions and the role of androgens in AI-resistance mechanisms. Results: AD-EDR cell lines showed increased growth and induction of estrogen-responsive genes rather than androgen-responsive genes by androgens or 3β-diol. Further analysis revealed increased expressions of HSD3B1 and reduced expression of androgen receptor (AR) in these cell lines. In parental MCF-7-E10 cells, ectopic expression of HSD3B1 or inhibition of AR resulted in adaptation to estrogen-deprived and androgen-abundant conditions. In coculture with stromal cells replicating the local estrogen production from androgen, AD-EDR cell lines showed AI resistance compared with parental MCF-7-E10 cells. Immunohistochemistry and real-time PCR analyses on 9 pairs of primary and recurrent tissue samples from AI-resistant breast cancer revealed the decrease of AR protein expression in all cases and increase of HSD3B1 mRNA expression in 5. Conclusion: In the present study, we successfully cloned two stable variants that show ER activity depending on androgen metabolites. Investigation of these cell lines suggested that the increased function of 3β-HSD type 1 and reduced function of AR contribute to AI resistance by enhancing the androgen metabolite-dependent growth and reducing the inhibitory effect of androgens. Our data of clinical samples suggest that this mechanism also acts as an AI-resistance in clinical breast cancer in some cases. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-17.

Background: Endoxifen and 4-hydroxy tamoxifen are both active metabolites of tamoxifen and exert anti-tumor activity for ER+ breast cancer. However, only small numbers of paper reported biological profile of endoxifen. Methods: Cells were incubated with various concentration of endoxifen ( (Z)-4-Hydroxy-N-desmethyl Tamoxifen ( Results: IC50s of endoxifen for MCF-7 were 100 nM in estradiol (E2) deprivation condition and 500 nM in the presence of 1nM E2. These are significantly higher than those of 4-OH tamoxifen, which are 10 nM without E2 and 50 nM with E2, respectively. From the previous reports, plasma endoxifen concentrations in breast cancer patients were ranged from 20 to 200 nM according to CYP2D6 phenotype. Anti-proliferative index of 20 nM endoxifen for MCF-7 was 0.7 (indicating 30% reduction in proliferative activity), and 200 nM endoxifen was 0.5 in the absence of E2. These were 0.95 (20 nM) and 0.75 (200nM), respectively, when incubated with 1 nM E2. 10 nM 4-OH tamoxifen, which is mean concentration in patient plasma, showed about 0.6 anti-proliferative index with no regards to E2 condition. Without E2, endoxifen reduced ERα expression as previously reported, however, endoxifen increased ERα amount in the presence of E2 accompanied with decrease of HER-2 expression. This alternation of ERα and HER2 expression were identical to those observed using 4-OH tamoxifen. There was a weak tendency that ERβ expressing MCF-7 clones showed favorable response to endoxifen compared to parent MCF-7, but this was not significant. Conclusion: Endoxifen is active agent for MCF-7 cell, however, it requires 10-fold higher concentration compared to 4-OH tamoxifen to achieve IC 50. Difference of anti-proliferative effect between 20 nM and 200 nM of endoxifen are relatively small, and it might result in minor difference for clinical response, as expected from recent clinical studies of CYP2D6. This is the first time to show that endoxifen has opposite effect to ERα protein level depending on the presence or absence of E2. ERβ expression did not enhance activity of endoxifen in contrast to previous report. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-20.

The authors point out that air-core superconducting power transformers have the potential of serving not only as power transformers but also as shunt reactors when used in power transmission systems having large capacitances. Here, the rated primary volt-amperes, volt-amperes as a shunt reactor, and related quantities are analyzed and their relations to the magnetic coupling factor and magnetizing reactance of the transformer are clarified. >

In AKR/J mice inoculated intraperitoneally with protoscoleces of Echinococcus multilocularis, the oral administration of alpha-difluoromethylornithine (DFMO) in drinking water had no obvious effect on the development of hydatid cysts. The DFMO treatment also caused no apparent depletion of three major polyamines (putrescine, spermidine, and spermine) in the cysts, although a slight reduction in the putrescine level was detected. The failure of DFMO treatment to reduce the growth of cysts in mice may result from the absence of ornithine decarboxylase in the parasite.

An ultrafast monolithic 8-bit DAC is designed and fabricated. To realize this DAC, a new high-speed conversion technique, referred to as the data multiplexing method, and a variation of the segmented DAC (J.A. Shoeff, 1979) for low glitch are developed. The DAC is fabricated with shallow-groove-isolated 3-/spl mu/m VLSI technology with peak f/SUB T/'s of 4.5 GHz. An experimental 8-bit DAC features a conversion rate of over 500 MHz, a full-scale settling time to 1% of 2 ns, rise/fall times of 0.6 ns, and a glitch energy of 20 ps-V without input latches or a deglitcher.

Capture, rearrangement and/or fragmentation of 1-(5-oxazolyl)-1-alkylidenes and 1-(5-isoxazolyl)-1-alkylidenes are described.

Streptococcus mutans was classified by the electrophoretic properties of glucosyltransferases (GTases) and fructosyltransferases (FTases). The cells of serotypes a, d and g did not release extracellular FTases, although those from other serotypes did. The enzymes from cells of serotypes d and g synthesized a good deal of insoluble polysaccharide compared with other serotypes. The enzymes were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polyacrylamide gel-isoelectric focussing (PAG-IEF). Gels were stained for their activity and protein content. Enzymes belonging to the same serotype gave the same specific pattern on both gels. The seven serotypes could be classified into the following four groups: serotypes d and g, serotype a, serotypes c, e and f, and serotype b. The results agree well with some previous reports based on other methods. The molecular weights of three GTase bands were 156K, 146K and 135K, and of four kinds of FTase bands were 108K, 95K, 80K and 76K. The isoelectric points of main enzymes were 4.25, 4.60, 5.00, 5.55 and 5.70. Those of FTases were 4.25 and 4.60.

A new method was developed for the assay of ornithine decarboxylase (ODC)-antizyme complex, in which alpha-difluoromethylornithine (DFMO)-inactivated ODC was used to release active ODC competitively from the complex. ODC-antizyme complex was present in the extracts of hepatoma tissue-culture (HTC) cells and of ODC-stabilized variant HMOA cells, in much larger amounts in the latter. Cellular amounts of the complex fluctuated after a change of medium in a similar manner in HTC and HMOA cells, increasing during the period of ODC decay. After treatment with cycloheximide, the decay of ODC-antizyme complex in HMOA cells was more rapid than the decay of free ODC, but it was much slower than the decay of free ODC or complexed ODC in HTC cells. Administration of putrescine caused a rapid increase in the amount of ODC-antizyme complex in both HTC and HMOA cells, but nevertheless the decay of total ODC (free ODC plus ODC-antizyme complex) was more rapid with putrescine than with cycloheximide. These results suggested the possibility that ODC is degraded through complex-formation with antizyme. In contrast with complexed antizyme, free antizyme was not stabilized in HMOA cells.