E2F-1 transcriptional activity is a critical determinant of Mdm2 antagonist-induced apoptosis in human tumor cell lines.

Nutlin-3 is a selective inhibitor of the p53-Mdm2 interaction, and inhibits growth in most tumor cells with wild-type p53. However, it only induces apoptosis in subsets of tumor cells. We report that the apoptotic response induced by Nutlin-3 correlates with its antitumor effects in xenograft models in athymic mice. We have investigated signals that sensitize cells to undergo apoptosis induced by Nutlin-3. We demonstrate that adenovirus E1A increases Nutlin-3-induced apoptosis through pRb inhibition in mouse embryonic fibroblast cells in a p53-dependent manner. Consistent with this, pRb depletion by siRNA transfection with Nutlin-3 synergistically increases apoptosis in HCT116 human colon cancer cells, which are insensitive to induction of apoptosis by Nutlin-3 alone. As pRb is a key negative regulator of E2F, we asked whether E2F transcriptional activity determines the apoptotic response of cancer cells to Nutlin-3. We demonstrate that transcriptional activity of E2F correlates with the apoptotic response to Nutlin-3 in various tumor cells and depletion of E2F-1 suppresses Nutlin-3-induced apoptosis in cells possessing high transcriptional activity of E2F, including retinoblastoma cells harboring mutated Rb with wild-type p53. Furthermore, we report that expression of the p53 homologue p73, a target of E2F-1, is markedly increased by Nutlin-3 in Rb-mutated tumor cells harboring wild-type p53. Depletion of p73 by siRNA transfection suppresses Nutlin-3-induced apoptosis in these cells. Taken together, our results demonstrate that E2F-1 transcriptional activity is a critical determinant of Mdm2 antagonist-induced apoptosis and p73 is important for E2F-1-mediated apoptosis induced by Nutlin-3, especially in tumor cells with mutated Rb. Furthermore, our results suggest that tumor cells, including Rb mutated cells, which harbor wild-type p53 and high E2F transcriptional activity, could be a good target for Mdm2 antagonist therapy.

Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression.

To elucidate the role of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, in tumour angiogenesis and malignant progression, an expression vector harboring human VEGF cDNA was stably transfected into three human cancer cell lines with poor VEGF productivity. Though their in vitro growth rate and intrinsic productivity of another angiogenic factor, basic fibroblast growth factor (bFGF), were not changed by transfection, those clones with higher VEGF production were endowed with tumorigenic and angiogenic potentials as follows: firstly, nontumorigenic, lung carcinoma QG90 cells having lower bFGF productivity acquired tumorigenicity as well as significant in vivo angiogenesis-inducing ability, secondly, tumorigenic colorectal carcinoma RPMI4788 cells having higher potency for bFGF production could form more vascularized solid tumour with faster growth rate and thirdly, oestrogen-dependent breast carcinoma MCF-7 cells, which did not produce detectable bFGF, acquired tumorigenicity even in the absence of oestrogen and the solid tumour growth rate was remarkably enhanced, accompanied with increased vascularization, in the presence of oestrogen. These results suggest that tumour progression closely depends on angiogenesis, and VEGF significantly contributes to malignant progression of a variety of tumour cells through its potent angiogenic activity, independent on the bFGF productivity of tumour cells.

Different antitumor activities of anti-bFGF neutralizing antibodies: heparin-binding domain provides an inefficient epitope for neutralization in vivo.

Basic fibroblast growth factor (bFGF) plays an important role in tumor growth and angiogenesis. To elucidate the efficient recognition sites by anti-bFGF neutralizing antibodies, we generated two anti-bFGF neutralizing monoclonal IgG1 antibodies (mAbs), 2G11 and 1E6, recognizing different sites, and estimated as binding to the heparin-binding and the receptor-binding regions of bFGF, respectively, both of which have been shown to be important for its receptor interaction. Despite their high in vitro anti-bFGF activity in the absence of heparin, 2G11, with in vitro activity in competition with heparin, failed to inhibit the in vivo tumor growth of bFGF-producing RPMI4788 cells, though 1E6, showing non-competition with heparin, exhibited a significant antitumor effect. These results show that the heparin-binding domain of bFGF provides an inefficient epitope for in vivo neutralization of anti-bFGF mAb, and anti-bFGF neutralizing mAbs without competition against heparin have the potential to show in vivo antitumor effects.

Tumorigenicity depends on angiogenic potential of tumor cells: dominant role of vascular endothelial growth factor and/or fibroblast growth factors produced by tumor cells.

The aim of this study was to determine the role of tumor-derived angiogenic factors in solid tumor formation. We compared the angiogenic potential of tumorigenic and non-tumorigenic human tumor cell lines. All tumorigenic cell lines induced angiogenesis in vivo and their angiogenesis-inducing abilities were higher than those of the other non-tumorigenic cell lines. This in vivo angiogenic potential was well correlated with the in vitro endothelial cell growth-stimulating activity contained in the cell extract or conditioned medium of each cell line. The endothelial cell growth-stimulating activities of these cell lines were completely inhibited by neutralizing antibodies to basic fibroblast growth factor (bFGF), acidic FGF (aFGF) or vascular endothelial growth factor (VEGF). Furthermore, the levels of tumor-derived endothelial cell growth-stimulating activities depended on the amounts of angiogenic factors such as VEGF and bFGF produced by tumor cells. Although VEGF transcripts were detected in all of the cell lines by RT-PCR assay, the non-tumorigenic cell lines showed poor productivity of VEGF as well as FGFs and had less or non-potency for endothelial cell growth stimulation. These findings suggest that the increase in production of angiogenic factors by tumor cells is necessary for their in vivo angiogenic and tumorigenic potentials, and that VEGF and FGFs are the major mediators of tumor-induced angiogenesis.

In vivo malignant phenotype of hst-1-transfected cells regulated by paracrine endothelial cell growth stimulation by HST-1.

The hst-1 gene product, one of the fibroblast growth factor family proteins, has transforming and angiogenic activities. The BALB/c 3T3 cell line was transfected with an expression vector harboring human hst-1 cDNA and the malignant properties of two stably transfected clones, TC-1 and TC-2, were examined. The stimulating activity of TC-1-conditioned medium for endothelial cell DNA synthesis was approximately four times stronger than that of TC-2-conditioned medium and correlated with hst-1 mRNA expression levels. Other than endothelial cell growth stimulation, these two clones had similar typical in vitro transformed properties, with identical doubling times and morphological changes. When nude mice were injected s.c. with these clones, TC-1 cells revealed faster tumor formation and growth, compared to TC-2 cells which had less potential to promote endothelial cell growth. Furthermore, the life span of mice injected i.v. with TC-1 cells was shorter than those with TC-2 cells, resulting from progressive tumor growth in the lungs. This advanced malignant in vivo behavior of TC-1 cells may be mediated by the high angiogenic potential of TC-1 cells secreting larger amounts of HST-1 compared to TC-2 cells, suggesting that angiogenesis contributes to malignant progression of tumors.

Flt-1 but not KDR/Flk-1 tyrosine kinase is a receptor for placenta growth factor, which is related to vascular endothelial growth factor.

Placenta Growth Factor (PIGF) is a new member of vascular endothelial growth factor (VEGF) family. Although VEGF binds Flt family Flt-1 and KDR/Flk-1 tyrosine kinases at high affinity for signal transduction, biological activities and the receptors of PIGF have not been extensively studied. Reverse transcription-PCR showed that PIGF-2, a subtype of PIGF-1 that bears a basic amino acid-rich domain, is more abundant than PIGF-1 and thus is the major subtype in human placenta. Using antibodies specific to PIGF-1 or -2 as markers, we obtained large amounts of PIGFs in the baculovirus expression system. PIGF-2 had growth-stimulatory activity on human umbilical vein endothelial cells and vascular permeability activity in the Miles assay at levels about 10-fold lower than those of VEGF. All PIGF-1 activities were lower than those of PIGF-2. Both PIGFs competed for the binding of 125I-labeled VEGF to Flt-1 receptor but not to KDR/Flk-1 expressed on NIH3T3 cells. Furthermore, 125I-labeled PIGF bound to Flt-1 at high affinity but not to KDR/Flk-1. Supporting the notion that PIGF can use only Flt-1 as a receptor, PIGF activated Flt-1 to autophosphorylate, whereas PIGF could not generate signals from KDR/Flk-1. These results indicate that Flt-1, but not KDR/Flk-1, is a receptor for PIGF, suggesting that the weak biological activities of PIGF are due to its use of only part of the available VEGF signaling. These mild characteristics of PIGF may be important for the appropriate development and maintenance of normal placental tissue.

Pharmaceutical and biological properties of doxorubicin encapsulated in liposomes (L-ADM): the effect of repeated administration on the systemic phagocytic activity and pharmacokinetics.

We investigated the biodistribution and antitumour activity of doxorubicin (ADM) encapsulated in liposomes (L-ADM) after two administrations in tumour bearing mice. The effect of the first administration on phagocytic activity was also examined. The biodistribution of L-ADM after the second dosing at an interval of 4d was remarkably different from that after the first. The concentration of ADM in plasma and tumour after the second injection was higher, but that in the liver was lower than after the first administration. This decrease in distribution to the liver is thought to have contributed to the difference in the biodistribution characteristics of L-ADM. With regard to antitumour effect, the activity was similar between L-ADM and a solution of ADM (F-ADM). To investigate the effect of the first administration of L-ADM on biodistribution, systemic phagocytic activity was measured after the injection of F-ADM, L-ADM, or 'empty' liposomes not containing ADM. F-ADM and L-ADM (7.5 mg ADM/kg body weight) reduced phagocytic activity to approximately 50% and 30% of control, respectively. This finding suggests that entrapment of ADM in liposomes enhances both the distribution of the drug to the reticuloendothelial system (RES) and its suppressive effect on RES activity. These results indicate that the decrease in RES activity by L-ADM must be considered in estimation of the pharmacokinetics, antitumour activity, and toxicity of L-ADM in clinical use when given by repeat administration or used in combination with other antitumour agents.

A new water-soluble camptothecin derivative, DX-8951f, exhibits potent antitumor activity against human tumors in vitro and in vivo.

CPT-11, a semisynthetic derivative of camptothecin, exhibited strong antitumor activity against lymphoma, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, and cervical cancer. CPT-11 is a pro-drug that is converted to an active metabolite, SN-38, in vivo by enzymes such as carboxylesterase. We synthesized a water-soluble and non-pro-drug analog of camptothecin, DX-8951f. It showed both high in vitro potency against a series of 32 malignant cell lines and significant topoisomerase I inhibition. The anti-proliferative activity of DX-8951f, as indicated by the mean GI50 value, was about 6 and 28 times greater than that of SN-38 or SK&F 10486-A (Topotecan), respectively. These three derivatives of camptothecin showed similar patterns of differential response among 32 cell lines, that is, their spectra of in vitro cytotoxicity were almost the same. The antitumor activity of three doses of DX-8951f administered i.v. at 4-day intervals against human gastric adenocarcinoma SC-6 xenografts was greater than that of CPT-11 or SK&F 10486-A. Moreover, it overcame P-glycoprotein-mediated multi-drug resistance. These data suggest that DX-8951f has a high antitumor activity and is a potential therapeutic agent.

Intracellular roles of SN-38, a metabolite of the camptothecin derivative CPT-11, in the antitumor effect of CPT-11.

It is known that 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), a semisynthesized derivative of camptothecin (CPT), has a potent antitumor activity in vivo, but 7-ethyl-10-hydroxycamptothecin (SN-38), a metabolite of CPT-11, shows much stronger cytotoxicity in vitro than CPT-11. In this study, we demonstrated that the relaxation of SV40 DNA plasmids by type I DNA topoisomerase prepared from P388 murine leukemia cells was inhibited by 50% by SN-38 at approximately 1 microM, although CPT-11 at 1 mM slightly inhibited the relaxation. SN-38 and CPT showed strong, time-dependent inhibitory activity against DNA synthesis of P388 cells. However, CPT-11 weakly inhibited DNA synthesis independently of time with coincident inhibition of the total thymidine uptake by the cells. By alkaline and neutral elution assays, it was demonstrated that SN-38 caused much more frequent DNA single-strand breaks in P388 cells than did CPT-11. The same content of SN-38 and a similar frequency of single-strand breaks were detected in the cells treated with SN-38 at 0.1 microM or with CPT-11 at 100 microM. Therefore, single-strand breaks by CPT-11 seem to be due to SN-38 produced from CPT-11 in cells. These results indicate that CPT-11 itself possesses a marginal antiproliferative effect but that SN-38 plays an essential role in the mechanism of action of CPT-11.

Antitumor activity of a camptothecin derivative, CPT-11, against human tumor xenografts in nude mice.

The antitumor effects of the camptothecin (CPT) derivative CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin , were tested on human tumor xenografts in nude mice. CPT-11 showed antitumor activity higher than that of Adriamycin, 5-fluorouracil, or futraful, with little or no reduction of body weight being observed in the mice. The growth of colon adenocarcinoma Co-4 was significantly inhibited after a single i.v. injection of CPT-11 at 25, 50, or 100 mg/kg. The single i.v. injection was also significantly effective against mammary carcinoma MX-1 and gastric adenocarcinoma St-15. All of the mice bearing MX-1 tumors were cured by the administration of CPT-11 every 4 days for a total of three treatments at a total dose of 200 mg/kg given i.v. or of 400 mg/kg given p.o. Three i.v. or oral treatments were also effective against Co-4, St-15, gastric adenocarcinoma SC-6, and squamous-cell lung carcinoma QG-56. To achieve the same efficacy attained by i.v. injection, however, oral doses 2-4 times higher than the i.v. doses were required. When the total dose was fixed at 100 mg/kg, a triple i.v. injection was most effective, followed by a single i.v. injection and, finally daily p.o. administration for 10 days. Although SN-38 (7-ethyl-10-hydroxycamptothecin), a metabolite of CPT-11, showed much stronger cytotoxic activity in vitro than did CPT-11, its antitumor effects were similar, if not inferior, to those of CPT-11 in vivo at the same dose level. CPT-11 was converted into SN-38 by human tumors, but the sensitivity of these tumors to CPT-11 in vivo was independent of their ability to produce SN-38. These results suggest that CPT-11 may be clinically effective, depending on the schedule of administration, but that its effectiveness is not related to the ability of the tumor to produce SN-38.

[Ultrasonic and biochemical detection and prenatal treatments of intra-uterine fetal growth retardation (author's transl)].

Efficacy of three ultrasonographic and six biochemical methods for the detection of intrauterine growth retardation were assessed in prospective studies of 40 cases associated with short uterine fundal height less than -1.5 SD and/or small ultrasonographically determined total intrauterine volume (TIUV) less than -1 SD of normal populations. Prenatal treatments, consisting of bed rest, high protein diet, intravenous drip infusion of 10% maltose, 500 ml per day, for more than 12 days, etc., were administered on them. Fifteen cases (37.5%) delivered small-for-date infants, 9 of which complicated by toxemia of pregnancy. At the final determinations, small TIUV were found in all small-for-date cases (100%), short biparietal diameter 80.0%, and short longitudinal intracavital uterine length 53.3% of 15 small-for-date cases. In biochemical parameters, low maternal plasma estriol levels were found in 73.3%, low plasma human placental lactogen levels 66.7%, low urinary estriol excretion 53.3%, abnormal plasma alpha-fetoprotein levels 33.3%, and low plasma progesterone levels 20.0% of 15 small-for-date cases. Nineteen cases (47.5%) demonstrated remarkable increases in TIUV following prenatal treatments, and delivered appropriate-for-date infants. Despite of marked growth in biophysical parameters, abnormal biochemical values were mostly not improved by these treatments.