DEVELOPMENT OF AN ADRIAMYCIN RESISTANT MURINE TUMOR S-180 CELL LINE IN VIVO

Adriamycin resistant cells were obtained from low dotage treated BABL/c mice Inoculated with S-180 cells. Resistance of these cells for adriamycin was 66-fold more than their parental cells. The resistance for a typical DNA topoisomerase Ⅱ inhibitor VP16 (Etopcaide) was increased 9 times. Overexpression of multidrug resistant gene (MDR gene) products, P-glycoproteins (P-1 70), was also demonstrated by immunohistochemistry. Furthermore, the ability of the resistant cells to reduce net cellular drug accumulation measured by flow fluorescence cytometry was 89-fold higher than their parental cells. These results support the hypothesis that the resistance of S-180R cells to adriamycin was mainly due to the overexpression of P-glycoproteins. The S-180R cells will be useful to select drugs or some other therapeutic strategies to overcome multidrug resistance in vivo.     

Differential expression of intermediate-filament proteins in murine sarcoma 180 ascites or solid tumor

The intermediate-filament proteins in Sarcoma 180 ascites cells and solid tumors generated by s.c. injection of ascites cells in NMRI or nude mice were analyzed by one- and two-dimensional gel electrophoresis and identified by immunological methods. The ascites form of Sarcoma 180 coexpresses keratin and vimentin, whereas the solid tumor ceases to synthesize keratins but continues to express vimentin. These reversible changes in the expression of intermediate-filament proteins may be due to a change in the differentiation program induced by environmental conditions like growth with or without cell contact.     

Optimization of high-dose methotrexate with leucovorin rescue therapy in the L1210 leukemia and sarcoma 180 murine tumor models.

An analysis of dose and schedule dependence of calcium leucovorin rescue during high-dose methotrexate therapy of ascitic forms of l1210 leukemia and Sarcoma 180 is reported. Schedules with very delayed "low-dose" leucovorin rescue following lethal doses of methotrexate were highly effective in preventing toxicity and achieved a pronounced antitumor effect in both ascites tumor models. Best results were obtained on a schedule of methotrexate (400 mg/kg s.c.) followed 16 to 20 hr later by calcium leucovorin (12 mg/kg s.c.) given once every 2 hr for a total of 5 doses. Progressive increases in the calcium leucovorin dosage on any schedule reduced both toxicity and the antitumor effect of methotrexate in each model. Following a single course of therapy, essentially no toxicity was observed, and the antitumor effects were 2-fold (L1210 leukemia) and 4-fold (Sarcoma 180) greater than a single, maximally tolerated dose (24/kg s.c.) methotrexate alone. An increase in the methotrexate dosage to 800 mg/kg s.c. with or without an increase in calcium leucovorin dosages on the same schedule did not appreciably increase the antitumor effect observed. Two courses of high-dose methotrexate (400 mg/kg s.c.) with leucovorin rescue (24 mg/kg s.c. 16, 20, and 24 hr after drug) given with an 8-day interval between courses doubled the total antitumor effect in each model with no substantial increase in toxicity and gave long-term survivors with Sarcoma 180. The results, overall, are in close agreement with prior prediction for schedule and dose dependence made on the basis of related pharmacokinetic and biochemical studies in murine tumor models reported from this laboratory.     

Comparison between in vitro radiosensitivity and in vivo radioresponse in murine tumor cell lines. II: In vivo radioresponse following fractionated treatment and in vitro/in vivo correlations

Survival in the low-dose region of in vitro radiation survival curves for human tumor cell lines may be correlated with the expected clinical radiocurability of tumors of similar histopathological type. The present investigation examined this hypothesis using a series of transplantable murine solid tumors. The in vivo radioresponse of the tumors following fractionated dose (10 fractions of 2 Gy given at 4 hr intervals) and single dose (20 Gy) radiation treatment was measured by growth delay and tumor cell survival assays. The measurements of tumor cell survival were initiated either immediately or 8 hr after the end of radiation treatment. There was no evidence for repair of potentially lethal damage in vivo in any of the tumors. The measurements of in vivo response were compared to parameters of in vitro radiation survival curves for the same tumor cell lines, which were measured concurrently. The tumor cell survival following 10 fractions of 2 Gy correlated best with the measured in vitro survival at 2 Gy, although good correlations were also observed with two parameters calculated from the in vitro data; the value of alpha from fitting the linear-quadratic (LQ) model and the mean inactivation dose (MID). Correlations were also observed between specific growth delay measured in vivo following 10 fractions of 2 Gy and these in vitro parameters. Despite the correlations observed, the measured survival values following 10 fractions of 2 Gy in vivo did not agree quantitatively with the theoretical values predicted from the in vitro survival data assuming equal effect for each fraction. This discrepancy indicates that other factors also contribute to the overall response of a tumor to fractionated irradiation. Nevertheless, these findings support the idea that intrinsic radiosensitivity plays a significant role in determining the overall response of a tumor to fractionated irradiation and provides strong support for the concept of testing the in vitro radiation sensitivity of biopsy specimens as a predictive assay of clinical radiocurability.     

Phase-specific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma pulmonary nodules.

The cytotoxic effects in vivo of hydroxyurea (HU) on murine fibrosarcoma (FSa) cells grown as pulmonary tumours were determined. Tumour cells from 13-day-old nodules were made into suspension and separated on the basis of cell size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations, as well as the degree of contamination by normal diploid cells in each of the tumour-cell populations. HU cytotoxicity was tested by administering both a single 1 mg/g i.p. dose into mice that had been injected i.v. 20 min earlier with known numbers of synchronized viable FSa cells, and i.p. doses of 1 mg/g each into mice bearing 13-day-old pulmonary nodules. In the latter experiments, animals were killed 1 h after the last dose, and the tumour nodules were excised and made into a single-cell suspension and elutriated. Known numbers of cells from each fraction were injected into recipient mice to determine survival. In both sets of experiments, cell killing by HU correlated with the percentage of S-phase cells. The treatment of 13-day-old pulmonary nodules with 3 doses of HU also depleted the (G2+M) phase tumour cells and increased the heterogeneity between tumour subpopulations, as determined by FMF analysis.     

In vivo antitumor activity of ZYD88, a Chinese herbal medicine, in animal models with S180 sarcoma and Ehrlich tumor

Cancer is a leading cause of death in the world. The continuous development of effective and nontoxic therapeutic agents is a major task in the battle of cancer. We report the in vivo effects of a Chinese herbal medicine, ZYD88, on the inhibition of tumor growth in an S(180) xenograft animal model and the improvement of animal survival in the Ehrlich tumor model. Oral administration of ZYD88 in mice with the xenograft S(180) sarcoma significantly inhibited tumor growth in a dose-dependent manner. Moreover, ZYD88 given to the animals with Ehrlich ascitic tumors by gavage significantly prolonged the life span compared with that of animals treated with saline. In both animal models, the effects of ZYD88 were comparable to those of a standard chemotherapeutic agent, cyclophosphamide, although it had few side effects. These results clearly demonstrated the in vivo anticancer activity of ZYD88 in two different animal models and suggest that ZYD88 is a potential agent for the clinical management of cancer and warrants further preclinical and clinical investigations.     

Effects of recombinant alpha-interferon-gelatin conjugate on in vivo murine tumor cell growth.

A recombinant human alpha-interferon A/D (IFN), also known to be effective in mice, was conjugated to gelatin with a water-soluble carbodiimide. The IFN-gelatin conjugate was much more efficient than free IFN in activating mouse peritoneal macrophages (M?) in an in vitro experiment to inhibit the growth of IFN-resistant subline cells (RR1) of murine fibrosarcoma. A single i.p. injection of the conjugate administered to normal mice was also more effective than one of free IFN in activating peritoneal M? and natural killer cells in peritoneal exudate cell and spleen cell populations. In the investigation on body distribution of the IFN-gelatin conjugate, an enhanced affinity to M? as well as a prolonged retention were observed in comparison with free IFN. An injection of the IFN-gelatin conjugate i.p. was more effective than one of free IFN in suppressing the in vivo growth of not only IFN-sensitive SS2 cells but also RR1 cells in the peritoneal cavity of mice, although RR1 cells were only susceptible to the indirect effect of IFN via host cells, in contrast to SS2 cells. In addition to an increased recruitment of M? to the peritoneal cavity in RR1-bearing mice receiving i.p. injection of the IFN-gelatin conjugate, these M? were activated to inhibit the in vitro growth of RR1 cells. These results indicate that the IFN-gelatin conjugate is a promising antitumor agent that is much more effective than free IFN. The dose of IFN in the conjugate required for exerting the antitumor effects is much lower than that of free IFN, which leads to a reduction of adverse effects of IFN.     

In utero tumor induction by murine sarcoma virus (Moloney) in the rat. I. Biological characteristics

Female R rats mated with an R male and inoculated in utero, after fetectomy, with murine sarcoma virus (Moloney), developed tumors. These tumors originated in the uterus and were of fetal origin. Intravenous or intraperitoneal injection of the virus induced similar tumors. Infectious virus could not be isolated from the tumor cells kept as transplantable lines or cultured in vitro. However, the presence of the MSV genome could be demonstrated by a direct rescue test. In non-pregnant rats treated in the same way no neoplasms were recorded. The results are discussed in the light of current theories on the special immunological fetal-maternal relationship.     

Sulforaphane induces cell cycle arrest and apoptosis in murine osteosarcoma cells in vitro and inhibits tumor growth in vivo

Sulforaphane (SFN), a naturally occurring isothiocyanate, is an attractive agent due to its potent anticancer effects. SFN suppresses the proliferation of various cancer cells in vitro and in vivo. In this study, we report that SFN inhibited the proliferation of cultured murine osteosarcoma LM8 cells. Twenty micromolar SFN completely inhibited the growth of LM8 cells and caused G2/M-phase arrest. SFN induced the expression of p21(WAF1/CIP1) protein causing the cell cycle arrest in a dose-dependent manner. SFN induced apoptosis which was characterized by the appearance of cells with sub-G1 DNA content and the cleavage and activation of caspase-3. We showed that SFN induced the growth arrest and up-regulated the expression of p21(WAF1/CIP1) protein in a p53-independent manner in human osteosarcoma MG63 cells. We found that intraperitoneal administration of SFN (1 or 2 mg, 5 times/week) significantly inhibited the growth of LM8 xenografts to <30% of the controls in a preclinical animal model without causing any toxicity. In osteosarcoma cells, our findings provide in vivo evidence for the efficacy of SFN against the advanced growth of tumor. We showed that SFN induces cell cycle arrest and apoptosis in osteosarcoma cells and inhibits tumor xenograft growth. Furthermore, SFN is a potent inducer of p21(WAF1/CIP1) in osteosarcoma cells. These results raise the possibility that SFN may be a promising candidate for molecular-targeting chemotherapy against osteosarcoma.     

Sarcoma-negative leukemia-positive transformed cell culture established from a murine sarcoma virus-induced rat bone tumor.

Inoculation of the Soehner-Dmochowski isolate of the Moloney strain of murine sarcoma virus (MSV), designated MSV-SD, consistently leads to the development of bone tumors in the susceptible New Zealand black (NB) rats. Two separate cell cultures have been established from 2 individual MSV-SD-induced NB rat bone tumors. Cells of 1 bone tumor culture, designated RBT-E, are in early in vitro passages. These cells form colonies in agar medium and take up 2-deoxy-D-[3H]glucose at a greatly enhanced rate, 5 times that of normal nontransformed rat embryo cells. Cells of the RBT-E culture release both MSV and murine leukemia virus (MuLV) and therefore contain sarcoma-positive leukemia-positive transformed cells. The other rat bone tumor culture, designated RBT-L, produced MSV at early passages. RBT-L culture has been passaged over 130 times in vitro. Cells of the RBT-L culture form colonies in agar medium and take up 2-deoxy-D-[3H]glucose at an enhanced rate (3 times that of rat embryo cells), indicating the presence of transformed cells within the RBT-L culture. However, cells of the RBT-L culture at late passages (Passage 130 or more) produce only MuLV and no detectable MSV activity (as shown by the lack of tumor-inducing activity and the lack of focus-forming activities by direct assay or by infectious center assay). Attempts to rescue MSV activity from RBT-L cells by cocultivation with MuLV-producing mouse cells were not successful. The MuLV found in the RBT-L cells, however, is a competent helper virus capable of rescuing the MSV genome from MSV-SD-induced hamster bone tumor cells. All the available evidence supports the notion that late passages of the RBT-L culture contain transformed cells that do not produce conventionally detectable MSV. These cells are referred to as sarcoma-negative leukemia-positive cells. The sarcoma-negative leukemia-positive cells represent a different kind of MSV-induced transformed cells and provide a unique system for studies in search of MSV markers such as MSV-specific antigens and MSV-specific nucleotide sequences.     

In utero tumor induction by murine sarcoma virus (Moloney) in the rat. II. Histological and ultrastructural characteristics

On histological examination the twelve MSV-induced tumors in the uterus observed in our study proved to be of fetal origin. Ten of the 12 tumors had the histological and ultrastructural characteristics of a yolk-sac tumor. The two other tumors examined had a different morphological structure. One was classified as a teratoma with embryonal carcinoma, the other as a carcinosarcoma. In the primary neoplasms virus particles were found. Their ultrastructure was similar to that of MSV. The histology and the electron microscopy results are discussed in relation to the morphogenesis and the occurrence of these tumors in rodents as well as in man.     

环磷酰胺治疗小鼠S180肉瘤的体内31P磁共振波谱研究

目的 观察环磷酰治疗小鼠Ｓ１８０肉瘤的＾３１ＰＭＲＳ参数变化,以及这些参数的变化是否早于常规观察的瘤体积变化。方法 利用表面线圈＾３１ＰＮＭＲ方法,并研究了小鼠皮下接种Ｓ１８０肉瘤的体积。结果：对照组的ＰＣｒ／Ｐｉ和＋β－ＮＴＰ／Ｐｉ比值降低,而环磷酰胺给药组升高,且给药后２４,４８,７２ｈ时显著高于对照组（Ｐ〈０．０５）,对照组的ＰＭＥ／β－ＮＴＰ比值继续升高,而给药组ＰＭＥ／β－ＮＴＰ比值在给     

Dynamics of tumor cell clonogen repopulation in a murine sarcoma treated with cyclophosphamide

Experiments were performed to establish the extent and kinetics of tumor cell repopulation in a murine sarcoma, designated SA-NH, treated with cyclophosphamide (CY). Mice bearing 8-mm leg tumors were treated with 200 mg/kg CY which caused a transient tumor regression. Changes in the absolute clonogen content of tumors was determined by the change in TCD₅₀ values (50% tumor control) obtained under hypoxic conditions of local tumor irradiation at different times after CY treatment until tumors regrew to the pretreatment size. For comparison, hypoxic TCD₅₀ values were determined during the growth of tumors not treated with CY. CY greatly depleted tumors of clonogenic cells as manifested by the reduction in the control TCD₅₀ value of 64.5 Gy to 32.8 Gy 1 day after CY treatment. The reduced TCD₅₀ value remained unchanged for 2 weeks after treatment with CY, at which time the TCD₅₀ began to rapidly increase, continuing until the end of the observation period of 21 days when tumors reached the pretreatment size. In contrast, there was a constant but slower increase in TCD₅₀ values during the growth of tumors not treated with CY. The daily increase in TCD₅₀ was more than twice as high in CY-treated than in CY-untreated tumors: 4.5 Gy/day versus 2.1 Gy/day. This implies that the rate of clonogen production in CY-treated tumors was twice as high as that of unperturbed tumors. The possibility that tumors regrowing after CY treatment might consist of cells cross-resistant to irradiation was rejected by experiments showing that tumors grown from cells taken from CY-recurrent tumors were not significantly more resistant than control tumors of the same size. Further studies revealed that systemic effects produced by CY are conducive to tumor cell proliferation but the contribution of this mechanism to the observed increased rate of tumor cell repopulation in the recurrent tumors is uncertain.     

Phase-specific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma cells synchronized by centrifugal elutriation.

The S-phase-specific cytotoxicity of hydroxyurea (HU) was tested on synchronized murine fibrosarcoma (FSa) cells lodged in the lungs of C3Hf/Bu mice. FSa cells from primary asynchronous cultures were separated and synchronized on the basis of size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations. After elutriation, 8000 viable FSa cells from each fraction, along with 10(6) heavily irradiated tumour cells (unseparated) were injected i.v. into whole-body-irradiated mice (20 per group). Under these conditions, 95% of the injected cells, regardless of size or position in the cell cycle, are arrested in the lungs. Twenty minutes later, hydroxyurea (HU, 1 mg/g) was administered i.p. into 10 animals of each group. Fourteen days later the animals were killed, their lungs removed and fixed, and the number of macroscopic tumour nodules counted. Killing of the initially injected cells by HU, as evidenced by a reduction in lung colonies in treated animals, correlated with the precentage of S-phase cells in each fraction. The greatest effect, an 80% reduction in colony number, was seen in Fraction 8, containing the largest percentage of S-phase cells (65%). These results demonstrate the usefulness of this procedure as a rapid method for characterizing the phase specificity of chemotherapeutic drugs in vivo.     

In vivo optical imaging of cancer cell function and tumor microenvironment

In vivo optical imaging using fluorescence and bioluminescence is superior to other methods in terms of spatiotemporal resolution and specificity, and represents a new technology for comprehensively studying living organisms in a less invasive way. Nowadays, it is an indispensable technology for studying many aspects of cancer biology, including dynamic invasion and metastasis. In observations of fluorescence or bioluminescence signals in a living body, various problems were caused by optical characteristics such as absorption and scattering and, therefore, observation of deep tissue was difficult. Recent developments in techniques for observation of the deep tissues of living animals overcame this difficulty by improving bioluminescent proteins, fluorescent proteins, and fluorescent dyes, as well as detection technologies such as two‐photon excitation microscopy. In the present review, we introduce these technological developments and in vivo application of bioluminescence and fluorescence imaging, and discuss future perspectives on the use of in vivo optical imaging technology in cancer research.     

Direct and indirect effects of interferon on in vivo murine tumor cell growth

We cloned two sublines (S1 and R1) of murine Meth A fibrosarcoma cells with respect to their sensitivity to a murine alpha/beta-interferon (IFN) preparation. The growth of S1 cells was suppressed and that of R1 cells was hardly affected by IFN in vitro. This was also the case with cells enclosed in cell-impermeable diffusion chambers in peritoneal cavities. Nevertheless, IFN suppressed the growth of not only S1 cells but also R1 cells in mice inoculated i.p. with these cells, and the survival rates of both S1 cell recipients and R1 cell recipients were markedly improved. S1 cells were observed microscopically to be injured by the direct effect of IFN in vitro and in vivo, but R1 cells in in vitro culture with IFN and those surviving in vivo in the presence of IFN appeared to proliferate well. In the peritoneal cavity of R1 recipients treated daily with IFN, the recruitment of macrophages was enhanced in comparison with untreated R1 recipients. Adherent peritoneal exudate cells obtained from IFN-treated, R1-bearing mice were highly suppressive for the in vitro growth of not only R1 cells but also allogeneic and human cells. The role of macrophages in the indirect effect of IFN on tumor cell growth is discussed.