Helga Naundorf

Characterization of two human mammary carcinomas, MT-1 and MT-3, suitable for in vivo testing of ether lipids and their derivatives

SummaryThe human mammary carcinomas MT-1 and MT-3 originate from surgical material and were transplanted in nude mice. Both tumors have been classified as estradiol- and progesterone receptor-negative. Therapeutic doses of hormones and anti-hormones remained without growth inhibitory effect. MT-1 and MT-3 proved to be sensitive to conventional cytostatic drugs used for treatment of mammary carcinomas; striking is their sensitivity to ether lipids. Therefore, they are considered suitable tumor models for this class of substances.

Development and characterisation of novel human multidrug resistant mammary carcinoma lines in vitro and in vivo.

Clinical chemotherapy of breast carcinomas must be considered insufficient, mainly due to the appearance of drug resistance. The multidrug resistance (MDR) phenotype, either intrinsically occurring or acquired, e.g., against a panel of different antineoplastic drugs, is discussed in relation to several MDR‐associated genes such as the MDR‐gene mdr1 encoding the P‐glycoprotein (PGP), the MRP gene (multidrug resistance protein) encoding an MDR‐related protein or the LRP gene encoding the lung resistance protein. Numerous experimental and clinical approaches aiming at reversing resistance require well‐characterised in vitro and in vivo models. The aim of our work was to develop multidrug resistant sublines from human xenotransplanted breast carcinomas, in addition to the broadly used line MCF‐7 and its multidrug resistant subline MCF‐7/AdrR. MDR was induced in vitro with increasing concentrations of Adriablastin (ADR) for several weeks, resulting in a 3.5‐ to 35‐fold increase in IC50 values using the MTT‐test. Cell lines were cross‐resistant toward another MDR‐related drug, vincristine, but remained sensitive to non‐MDR‐related compounds such as cisplatin and methotrexate. The resistance toward Adriamycin and vincristine was confirmed in vivo by a lack of tumour growth inhibition in the nude mouse system. Gene expression data for the mdr1/PGP, MRP/MRP and LRP/LRP on both the mRNA (RT‐PCR) and the protein levels (immunoflow cytometry) demonstrated that induction of mdr1 gene expression was responsible for the acquired MDR phenotype. Rhodamine efflux data, indicated by PGP overexpression, underlined the development of this MDR mechanism in the newly established breast carcinoma lines MT‐1/ADR, MT‐3/ADR and MaTu/ADR. Int. J. Cancer 72:885–891, 1997.

Establishment and characterization of a new human oestradiol- and progesterone-receptor-positive mammary carcinoma serially transplantable in nude mice

SummaryA human mammary carcinoma originating from a postmenopausal patient was successfully transplanted into nude mice. According to the adopted criteria the tumour proved to be oestradiol- and progesterone-receptor-positive. Histological studies of the patient tumour revealed a ductal invasive mammary carcinoma with 80% tubular growth pattern. Following transplantation the adenoid structures decreased to 30%; the mitosis rate and grade of malignancy increased. Treatment of the nude mice with 20 μg oestradiol benzoate/mouse caused a loss of the oestradiol receptor of the mammary carcinoma. The mammary carcinoma 3366 can be used for testing of antineoplastic substances, antihormones and for studies in regard to down-regulation or blocking of hormone receptors and possible consequences for therapies.

Development and characterization of a tamoxifen-resistant breast carcinoma xenograft

A human tamoxifen-resistant mammary carcinoma, MaCa 3366/TAM, originating from a sensitive parental xenograft 3366 was successfully established by treatment of tumour-bearing nude mice with 1–50 mg kg–1 tamoxifen for 3 years during routine passaging. Both tumours did not differ significantly in OR- and PR-positivity, however, when compared with the sensitive tumour line, the mean OR content of the TAM-resistant subline is slightly lower. An OR-upregulation following withdrawal of oestradiol treatment was observed in the parental tumours but not in the resistant xenografts. Following long-term treatment with tamoxifen, the histological pattern of the breast carcinoma changed. The more differentiated structures being apparent after treatment with 17β-oestradiol in the original 3366 tumour were not induced in the resistant line. Tamoxifen failed to induce a tumour growth inhibition in comparison to the tamoxifen-sensitive line. The pure anti-oestrogen, ICI 182 780, revealed cross-resistance. Sequence analysis of the hormone-binding domain of the OR of both lines showed no differences, suggesting that either mutations in other regions of the OR are involved in the TAM-resistance phenotype or that mechanisms outside of this protein induced this phenotype. Oestrogen and anti-oestrogen regulate pS2 and cathepsin D expression in 3366 tumours as in the human breast cancer cell line MCF-7. The resistant 3366/TAM tumours have lost this regulation. The established breast cancer xenografts 3366 and 3366/TAM offer the possibility of investigating mechanisms of anti-oestrogen resistance in an in vivo situation. They can be used to test novel approaches to prevent, or to overcome, this resistance in a clinically related manner.

Establishment and characteristics of two new human mammary carcinoma lines serially transplantable in nude mice

Two human mammary carcinomas of postmenopausal women were successfully transplanted into nude mice. Both tumours were classified as epidermal-growth-factor-, oestradiol- and progesterone-receptor-negative and c-erbB2-protein-positive. Histological studies of the primary tumours (4000 and 4151) revealed ductal invasive mammary carcinomas. In the first passages the precondition for the growth of breast carcinoma 4000 were pretreatments of the nude mice with oestradiol and peanut oil before transplantation. The mammary carcinomas 4000 and 4151 described here are suitable for in vivo testing of antineoplastic substances and for biological studies.

Establishment and characteristics of two new human mammary carcinoma lines in nude mice with special reference to the estradiol receptor status and the importance of stroma for in vivo and in vitro growth.

SummaryTwo new human mammary carcinoma lines originating from surgical material were established in nude mice. According to the adopted criteria, the tumor 4049 has been classified as estradiol receptor positive and mammary carcinoma 4296 as estradiol receptor negative. Both tumors proved to be c-erbB-2 protein positive and EGF-receptor negative. In contrast to carcinoma 4296, thein vitro growth and the take rate of mammary carcinoma 4049 in nude mice seems to be dependent on stromal components. Pretreatment of mice with estradiol/peanut oil before tumor engraftment was an essential precondition for the growth of the primary tumor in nude mice. After successful establishment the tumor growth was significantly stimulated by estradiol. The growth rate of mammary carcinoma 4296 was independent of any supplementation of estradiol. The two breast tumors were characterized with regard to their growth behaviour, histology, and sensitivity to cytostatics and antihormones. They are considered suitable tumor models for the testing of antineoplastic substances and for biological experiments.

Relation of oestradiol-mediated growth stimulation with the expression of c-erbB-2 protein in xenotransplanted oestradiol-receptor-positive and -negative breast carcinomas.

Attempts were made to correlate growth effects induced by oestradiol and tamoxifen with the hormonal regulation of c-erbB-2 protein in experiments in vivo. We report here the responsiveness of four xenotransplanted oestrogen-receptor(ER)-positive and four ER-negative human mammary carcinomas to oestradiol and tamoxifen. Oestradiol in a dose of 0.5 mg/kg significantly increased the growth of the ER-positive mammary carcinomas 3366, MCF-7, 4134 and 4049, but not the ER-negative tumours 4000, 4296 and MT-3. However, within the group of the ER-negative breast carcinomas the tumour 4151 ES deviates from this growth behaviour, as we could prove an estrogen induced growth. The stimulation of tumour growth by oestradiol was always accompanied by a down-regulation of c-erbB-2 protein both in the ER-positive mammary carcinomas and in the ER-negative mammary carcinoma 4151 ES. Tamoxifen significantly inhibited the growth of the ER/PR-positive mammary carcinomas 3366 and MCF-7 but not the ER-positive/PR-negative mammary carcinomas 4049 and 4134. In the group of ER-negative mammary carcinomas only the growth of the oestrogen-responsive tumour 4151 ES was significantly inhibited by tamoxifen. The inhibition of tumour growth by tamoxifen was correlated with a reversion of the oestradiol-induced down-regulation of c-erbB-2, also in the ER-negative/oestradiol-responsive mammary carcinoma 4151 ES. From our results we hypothesize that the oestrogen-dependent growth of ER-negative breast carcinoma 4151 ES could also be correlated with the oestradiol-regulated expression of c-erbB-2 protein.

Carboplatin-liposomes (CPL) in immunodeficient mice: Improved antitumor activity for breast carcinomas and stimulation of hematopoiesis

Carboplatin-liposomes (CPL) have been shown to possess a strong stimulatory activity on the hematopoiesis in immunocompetent mice. As we were interested in studying this pharmacological characteristic in parallel with any antitumour effects which might be expected for the encapsulated cytostatic, we used a panel of six human breast carcinomas xenotransplanted to nude mice. The antitumor activity as well as the hematopoietic effects of the vesicles were studied in comparison to, and in combination with, the free drug. Carboplatin was encapsulated into reverse phase evaporation vesicles (REV) and injected i.p. as a single dose of 75 mg kg-1 into tumor-free and breast-carcinomabearing animals, respectively. Following a single application of CPL in nude mice, a significant increase of the WBC numbers to about three times for that of the normal level could be observed over a period of at least 28 days. The elevation was due to an increase in both circulating granulocytes and lymphocytes. The peripheral effect was accompanied by a relative decrease of spleen cellularity, while the number of bone marrow cells was hardly affected. There was no influence detectable on circulating blood cells in SCID mice. However, a rather high toxicity of CPL for this immunodeficient mouse strain was noticed. In the panel of breast carcinomas used, free carboplatin and CPL displayed a different pattern of therapeutic efficiencies. In four of the five tumor models tested, a combination of the free with the liposomal drug showed a significant inhibition of tumor growth while effectively preventing a drug-induced ieukopenia. As a result, CPL may be recommended in tumor therapy protocols for future clinical trials in order to relieve a cytostatic-induced bone marrow suppression.

Differences in immunoreactivity of estrogen receptor (ER) in tamoxifen-sensitive and -resistant breast carcinomas: preclinical and first clinical investigations

Inherited or acquired tamoxifen resistance is a major constraint in the endocrinological treatment of breast carcinomas. We developed an enzyme-immunoassay that discriminates between tamoxifen-sensitive and -resistant tumors. The procedure was established and standardized using two xenografted breast carcinomas – 3366 (highly sensitive to tamoxifen) and 3366/TAM (acquired tamoxifen resistance). The latter model was developed by treatment of 3366 tumor-bearing nude mice during serial passaging over 3 years with tamoxifen. Both lines were estrogen receptor (ER) positive (101 or 82 fmol/mg protein), and revealed no differences in the nucleotide sequences of the hormone binding domain of the ER protein. However, while in the sensitive tumors an upregulation of ER levels was registered after estradiol treatment of tumor bearing nude mice, the ER expression in the resistant line remained unchanged. The tamoxifen sensitive and -resistant breast carcinoma 3366 differed, additionally, in their immunoreactivity of ER to mAB H222. While an incubation with estradiol or tamoxifen of immobilized ER prepared from cytosols of the sensitive tumors 3366 led to a significant increase in immunoreactivity, samples of resistant tumors failed in the exposition of additional immunologically reactive epitopes. These results were the basis for the development of an assay for determination of the tamoxifen response in patients. Our retrospective results with 38 breast tumors from a tumor bank indicated that patients with an increase of immunoreactivity of ER more rarely had a recurrence while under going tamoxifen therapy compared with patients expecting no increase. However, the data indicate interesting changes occurring with the ER of tam-resistant tumors that are to be explained by further mutational or protein-chemical analysis.

IN VIVO EVALUATION OF A DRUG-INDUCIBLE VECTOR SYSTEM FOR THE COMBINED GENE­ AND CHEMOTHERAPY OF CANCER*

Regulated expression of therapeutic genes in cancer gene therapy is used in a growing number of gene therapy approaches. Numerous conditionally active vectors have been tested carrying promoters which respond to certain factors or therapeutic modalities, such as irradiation, hyperthermia etc. [1].