Effect of five antineoplastic agents on tumor xenografts with different growth rates.

The effects of cyclophosphamide (Cy), doxorubicin (Dx), cisplatin (DDP), melphalan (L-PAM), and vincristine (VCR) on various human and animal tumor lines with different growth rates, growing as xenografts in NMRI (nu/nu) mice, were studied. Two types of response were observed: For Cy and Dx, the response of the xenografts was negatively correlated with tumor volume doubling time (TD), indicating that rapidly growing tumors were more sensitive to these drugs than were slowly growing tumors. For DDP, L-PAM, and VCR, the effects were positively correlated with the TD, indicating that slowly growing tumors were more sensitive to these drugs than rapidly growing tumors. The data are discussed in relation to the effects of the drugs on proliferating and nonproliferating cells obtained with other cell lines.

Polysomal polyadenylated RNA and albumin messenger RNA in Mastomys liver and in a chemically induced hepatocellular carcinoma.

A hepatocellular carcinoma line (H78) was established from a primary liver tumor induced in Mastomys natalensis by a single administration of dimethylnitrosamine. Six to 8 months after transplantation (passages 5 to 7), well-differentiated tumors, still containing glucogen-storing cells, were isolated and used for the preparation of RNA. Polysomal polyadenylated RNAs from Mastomys liver and from H78 tumor line were then compared by hybridization kinetics. Total kinetic complexities were 6.6 X 10(9) and 6.3 X 10(9) daltons for liver and tumor, respectively. Complexities of the high and middle abundant class were reduced in the hepatoma. Heterologous hybridization reactions revealed that, in terms of RNA mass, all or most of the polysomal polyadenylated RNA present in the liver was also present in the tumor and vice versa. However, shifts in the relative abundance of messenger RNA sequences were detected. In contrast to most other transplanted hepatomas, H78 has approximately the same content of albumin messenger RNA on its polysomes as has untreated liver.

Therapy of solid Walker carcinosarcoma 256 with bleomycin after synchronization with hydroxyurea.

Rats with the solid Walker carcinosarcoma 256 were synchronized with hydroxyurea (6 x 50 mg/kg or 1 x 300 mg/kg body weight) and treated with bleomycin (32 mg/kg body weight) at different time points thereafter. Bleomycin clearly affects Walker carcinosarcoma cells at the G1/S boundary or in S-phase. The improvement in the results of bleomycin therapy after pretreatment with hydroxyurea can mainly be accounted for by the synchronization of the tumour cells.

Therapeutic response of human lung tumour xenografts to cyclophosphamide.

The effect of cyclophosphamide, given as a single i.p. injection (240 mg/kg) on 13 various fast growing human lung tumours, transplanted and passed serially in athymic nude mice, were studied. Cyclophosphamide showed activity against all tumours studied. The sensitivity of the different tumour lines tested varied considerably. The faster a tumor was growing, the more pronounced was the inhibitory effect of cyclophosphamide on growth, independent of the histological type. If different lung tumours were treated at the same size and site (8-10 mm, s.c. right anterior flank), growth rate was a factor of decisive importance for the sensitivity of a tumour to cyclophosphamide.

Flow cytometric analysis of primary lung carcinomas and their lymph node metastases.

Specimens of primary lung carcinomas and lymph node metastases from the same 18 patients were investigated by means of flow cytometry. The number of DNA stemlines, DNA indices, the proportion of diploid cells in the tumors and the distribution of the cell cycle phases were compared. In 10 patients DNA stemlines and DNA indices were identical in primary tumors and metastases. In two cases the DNA indices were doubled in metastases. In 6 cases the primary tumors contained two abnormal DNA stemlines and their metastases contained only one aneuploid stemline. Gross differences between primary tumors and lymph node metastases with regard to the proportion of cell cycle phases could not be found. The large variation between primary tumors and lymph node metastases with regard to DNA stemlines indicates that flow cytometric analysis of lymph nodes gives only limited information about the primary tumors.

Relevance of DNA-fluorimetry and short-term resistance testing for adjuvant treatment of non-small cell lung carcinomas.

In a clinical study 127 patients with previously untreated stage III non-small cell lung carcinomas (NSCLC) were investigated using flow cytometry and an in vitro short-term test for predicting resistance to cytostatic agents. Patients with aneuploid tumors and tumors with high proliferative activity had significantly shorter survival times than those with diploid or low proliferating tumors. The aim of this study was to find out whether groups of patients classified according to the additionally observed prognostic factors, experience an advantage or disadvantage from particular modalities of treatment. Seventy-nine patients had surgery alone, 18 patients were treated additionally with chemotherapy, and 30 patients with radiation. Patients with aneuploid, low proliferating and in vitro resistant tumors showed no different survival rates after treatment with chemo- and radiotherapy adjuvant to surgery. In contrast, patients with high proliferating tumors died earlier under adjuvant chemotherapy and radiation. Patients with in vitro chemosensitive tumors had shorter survival times after irradiation than patients who had surgery alone or who were treated with adjuvant chemotherapy.

Sensitivity of human non-small cell lung cancer xenografts to cyclophosphamide and cisplatin.

In order to establish the usefulness of the nude-mouse human tumour xenograft system as a predictive screen for antineoplastic agents, the antitumour activity of cyclophosphamide (CTX) and cisplatin (DDP), two clinically active drugs, was tested against a panel of 14 human non-small cell lung tumour xenografts and the experimental results were compared with the clinical results reported in the literature. The poor clinical response of non-small cell tumours was reflected in the lack of response of the xenograft tumours to these two agents.

Experimentally induced infection with autonomous parvoviruses, minute virus of mice and H-1, in the African multimammate mouse (Mastomys coucha).

To determine whether the multimammate mouse (Mastomys coucha) could be used to evaluate rodent parvovirus-based vectors, neonates were subcutaneously inoculated with minute virus of mice (prototype strain, MVMp) or rat parvovirus H-1. The course of infection with both viruses was similar. Seroconversion occurred within two weeks after virus inoculation, as detected by use of hemagglutination-inhibition assays, and antibody titers remained high for the entire observation period of 12 months. Viral DNA and infective virions were detected in several organs of inoculated animals prior to seroconversion, as measured by use of Southern blotting and plaque assays, respectively. Infective particles subsequently became undetectable, whereas viral DNA imprints persisted in distinct organs for at least nine months. Clinical signs of parvovirus infection appeared around six weeks after virus inoculation, and consisted of hemorrhages, stunted growth, and transient hair color changes. Sudden death occurred in a significant fraction of animals infected with MVMp, but not H-1 virus, at the time of weaning. Altogether, MVMp, which is innocuous to its natural host, the mouse, and H-1 virus, which is poorly pathogenic to the rat, appear to be pathogenic for Mastomys coucha.

[Angiogenesis of human breast cancers: neovascularization in xenotransplants and oxygenation in situ in patients]. / Angiogenese von menschlichen Mammakarzinomen: Neo-Vaskularisation in Xenotransplantaten und Oxygenierung in situ bei Patientinnen.

OBJECTIVE: Recent reports have shown that vascularization of breast cancers is an independent prognostic parameter. Tumor growth and neovascularization were investigated in xenotransplants of breast cancers and tissue oxygenation was measured in patients with breast tumors. METHODS: Tumor cells of the breast cancer MX-1 were implanted intradermally in 40 nude mice. Tumor growth and neovascularization were quantified microscopically. Tissue oxygenation was determined in 66 patients with breast tumors using the pO2-histography technique. RESULTS AND CONCLUSION: The angiogenesis begins prior to tumor growth, is insufficient and leads to chaotic blood flow. This results in a heterogeneity of tissue oxygenation in breast cancers. Oxygenation at the tumor periphery appears to be of prognostic importance for clinical outcome of breast cancer.

[Failure to partial synchronization with hydroxyurea to improve results of chemotherapy of solid neurosarcoma in the rat (author's transl)]. / Keine Verbesserung der Chemotherapie beim soliden Neurosarkom durch Teilsynchronisation mit Hydroxyharnstoff

Partial synchronization of the slow-growing cell of solid neurosarcoma in the rat can be achieved by blocking DNA synthesis with hydroxyurea. Possible augmentation after synchronization of the therapeutic effects achieved with various cytostatics was tested. The substances were administered to tumour-bearing rats at various times after synchronization (at the transition from G1 to S phase; at maximal DNA synthesis; at the transition from the S to the G2 phases; at mitosis) and therapeutic effects on tumour gs. Cyclophosphamide and adriamycin reduced tumour size, the effect being dose-dependent. No differences were noted between synchronized and non-synchronized cells. Hydroxyurea and vincristine had significantly less effect on the tumour cells, this minimal effect not being enhanced by synchronization. The reported results were obtained in tests on 360 rats and are statistically significant (double variance analysis).

Activation of latent papillomavirus genomes by chronic mechanical irritation.

The skin of animals of a laboratory strain of Mastomys natalensis carrying endogenous, latent papillomavirus genomes was irritated by scratching with glasspaper. Hyperproliferation of the epidermis and amplification of viral DNA followed this treatment, and in approximately 27% of the animals virus-producing papillomas were induced.

[Correlation of in vitro testing and therapeutical results in animal transplanted tumors after cytostatic treatment]. / Korrelation von In-vitro-Testung und Therapieergebnis bei tierischen Transplantationstumoren nach Behandlung mit Zytostatika

The antineoplastic activity of 5 substances was tested in vivo and in vitro on four different tumours (plasmocytoma and melanoma Fortner III of the Syrian golden hamster, the Walker carcinosarkoma 256 and an adenocarcinoma of the rat). The substances involved were 2,3,5-triethyleniminobenzoquinone-(1,4) (triaziquone), actinomycin D, podophyllinic ethylhydrazide (mitopodozide), bleomycin and adriamycin hydrochloride. The effect of the substances in vivo was measured on the size of the tumour, and in vitro on the incorporation of 3-H-thymidine and 3-H-uridine in short-term incubations of tumour-cell suspensions. No correlation was observed between the 3-H-thymidine incorporation in vitro and the response of the tumours in vivo. On the other hand, the 3-H-uridine incorporation in the tumour-cell suspensions in vitro was in good agreement with the results of therapy in the animal experiments. This is compatible with the results of earlier experiments using other substances to investigate the possible correlation between tumour therapy and in vitro tests.

Studies on the specificity of tissue supernatants on the proliferation of liver in baby-rats.

Supernatants (105,000 g) prepared from extracts of liver and kidney were injected at different concentrations into 10 day-old rats and the resulting effects on 3H-thymidine incorporation in liver, kidney and lung were measured over a period of 24 hours. Incorporation in all three organs is inhibited by supernatants from both liver and kidney, with maximal activity between 12 and 18 hours after injection. Fractions prepared from liver supernatant by ultrafiltration and alcohol precipitation also showed no specific effect on liver cells. In order to test whether the activity of any factors in the supernatants is cell-cycle dependent, organs of baby rats were synchronized by two different procedures: a) injection of a 3.5% casein solution, b) injection of hydroxyurea (2 x 10 mg/kg body weight with an internal of one hour). Inhibitory effects were only observed when the supernatants were injected during the G1-phase of the target cells. Supernatants prepared in a similar way from other organs such as spleen, lung or heart also inhibited thymidine incorporation in casein-stimulated liver from baby rats. All of our investigations have therefore failed to find any indication of tissue-specific activity in these extracts.

[Irradiation of solid Walker carcino-sarcomas after synchronisation with hydroxyurea (author's transl)]. / Strahlentherapie solider Walker-Karzinosarkome nach Synchronisation mit Hydroxyharnstoff.

Sprague-Dawley rats with solid Walker carcino-sarcomas were synchronized with hydroxyurea (HU; 6 x 50 mg and 1 x 300 mg HU/kg body weight) and then irradiated at different time points (60Co). The synchronized tumors showed a significant delay of growth when irradiation was applied in the late G1 phase, at the transition G1/S and in the early S phase. The remaining phases of the cell cycle, especially the S phase showed the same sensitivity as the non-synchronized controls. Improvement of therapy by irradiation after HU application was largely due to the synchronization of tumor cells. Only the increased therapeutic effect of irradiation shortly after application of HU can be explained also by combination of both HU and irradiation.

Detection of induced tumour-resistance to cyclophosphamide by the in vitro short term test.

Treatment of the solid Walker carcinosarcoma of the rat for a period of one year with cyclophosphamide (40 mg/kg at each passage; tumours were transplanted weekly) resulted in the formation of a cyclophosphamide-resistant tumour cell line. Without further treatment, animals injected with non-treated or cyclophosphamide pre-treated cells survived for 10 days on an average. After therapy with cyclophosphamide (2 x 38 mg/kg), rats with non-treated tumour cells survived 22 days whereas those with pre-treated tumours survived only for 17 days. Tumour cells which were shown to be sensitive in vivo also exhibited a larger reduction in 3H-thymidine and 3H-uridine incorporation in the in vitro short term test after incubation with urine from cyclophosphamide-treated rats or with 4-hydroperoxy-cyclophosphamide. The resistance to cyclophosphamide which was detected in animal experiments with Walker carcinosarcoma can therefore also be observed using the in vitro short term test.

Tumour induction in the rodent Mastomys natalensis by activation of endogenous papilloma virus genomes.

Specific DNA sequences from human papillomavirus have recently been detected in carcinomas from epidermodysplasia verruciformis patients, and in vulvar and cervical carcinomas but the role of papilloma viruses in the aetiology of these tumours is unclear. Indeed, little is known about the mechanisms that convert benign papillomas into malignant tumours and it is not even possible in tumour induction. Here, we describe an animal system that permits an analysis of the interaction of papilloma virus genomes with carcinogenic agents at the molecular level. In our colony of Mastomys natalensis (a close relative of the rat family), we have found extrachromosomal papilloma virus genomes persisting in a variety of tissues such as skin, muscle, liver and colon. With the ageing of the animals, the average copy number of viral DNA in skin cells increases and virus-producing tumours begin to appear in Mastomys at about 1 year old. This process is drastically enhanced by chronic treatment with a tumour promoter and transcription of the viral genomes has been found to be correlated with tumour formation.

Sensitivity tests of tumors to cytostatic agents. I. Comparative investigations on transplanted tumors in vivo and in vitro.

With series of transplanted tumors, the activities of different cytostatic agents which directly influence the metabolism of nucleic acids (Actinomycin D, adriamycin, daunomycin, 5-fluorouracil, procarbazine, trenimon) was measured by determining 3-H-uridine incorporation in short-term (3hrs) incubations of tumor cell suspensions. Data obtained could be used to predict the response of each tumor to particular cytostatic agents in vivo. The activities of the cytostatic agents as determined using long-term tissue cultures (time of exposure of tumor cells to cytostatic agent 48 hrs were comparable to those obtained with the short-term test. In long-term cultures, determination of cell numbers gave results similar to those obtained by morphological evaluation. In SHORt-term test, differing sensitivities of tumors to cytostatics could be detected.

Chemical carcinogenesis by the two-stage protocol in the skin Mastomys natalensis (Muridae) using topical initiation with 7,12-dimethylbenz(a)anthracene and topical promotion with 12-0-tetradecanoylphorbol-13-acetate.

The long-term (34 weeks) topical administration of 7,12-dimethylbenz(a)anthracene (DMBA) to the skin of male and female Mastomys induced a broad spectrum of benign and malignant tumors in all animals treated. In a two-stage carcinogenesis experiment with topical initiation with DMBA and topical promotion with TPA, 50% of the animals developed both benign and malignant skin tumors. In general, benign tumors occurred between weeks 15 and 25, whereas malignant tumors were seen 40 weeks after initiation. In contrast to the situation in Mus musculus, the benign tumors consisted mainly of keratoacanthomas instead of fibroepitheliomas. In the non-initiated, TPA-treated, control group four benign and four malignant tumors were seen, whereas animals of the DMBA-initiated, acetone-treated control group were free of tumors. The promotion of virus transformed cells with TPA is discussed.

[Studies in vivo and in vitro on the influence of 5-fluorouracil and n'-(2'-furanidyl)-5-fluorouracil on the walker carcinosarcoma 256 in the rat (author's transl)]. / In vivo- und in-vitro-untersuchungen am soliden walker-karzinosarkom 256 der ratte nach einwirkung von 5-fluoruracil und n'-(2'-furanidyl)-5-fluoruracil.

We compare the effects of 5-fluorouracil and N'-(2'-furanidyl)-5-fluorouracil (Ftorafur) in vivo (tumor size) and in vitro ([6-3H]-deoxyuridine incorporation) on solid Walker carcinosarcoma of the rat. 5-Fluorouracil and Ftorafur have a dose-dependent effect on tumor size and on [6-3H]-deoxyuridine incorporation in vitro. The in vivo effects on tumor size are similar when the dose of Ftorafur calculated by its molecular weight is about 3 times higher than that of 5-fluorouracil. In vitro an about 650 times higher dose of Ftorafur is required to achieve a comparable reduction of [6-3H]-deoxyuridine incorporation in the tumor cells.