Influence on antiproliferative activity of structural modification and conjugation of gonadotropin-releasing hormone (GnRH) analogues.

The effect of various GnRH analogues, and their conjugates on proliferation, clonogenicity and cell cycle phase distribution of MCF-7 and Ishikawa human cancer cell lines was studied. GnRH-III, a sea lamprey GnRH analogue reduced cell proliferation by 35% and clonogenicity by 55%. Structural modifications either decreased, or did not alter biological activity. Conjugation of GnRH analogues including MI-1544, MI-1892, and GnRH-III with poly(N-vinylpyrrolidone-co-maleic acid) (P) through a tetrapeptide spacer GFLG(X) substantially increased the inhibitory effect of the GnRH analogues. The conjugate P-X-GnRH-III induced significant accumulation of cells in the G2/M phase; from 8% to 15.6% at 24 h and 9.8% to 15% at 48 h. It was concluded that conjugation of various GnRH analogues substantially enhanced their antiproliferative activity, strongly reduced cell clonogenicity and retarded cell progression through the cell division cycle at the G2/M phase.

Effects of methylacetylenic putrescine, an ornithine decarboxylase inhibitor and potential novel anticancer agent, on human and mouse cancer cell lines.

Sensitivity of several human and mouse cancer cell lines to methylacetylenic putrescine (MAP) was evaluated using clonogenic, sulforhodamine B and cell counting assays. The effects of MAP on cell morphology, cell cycle phase distribution and changes in polyamine metabolism of xenografted MCF-7 and MDA-MB-231 human mammary tumor cells were also investigated. On the basis of IC50 values, BHT-101 human thyroid carcinoma cells were the most sensitive (9 micrograms/ml), followed by P388 mouse lymphoma (32 micrograms/ml), MCF-7 (48 micrograms/ml) and MDA-MB-231 (110 micrograms/ml) human breast carcinoma cell lines. MAP treatment led to accumulation of P388 cells in G1 phase. At higher doses, the cytoplasm of the cells became vacuolated followed by apoptosis. The foamy cytoplasm may suggest a rare type of cell death (Clarke III type) called non-apoptotic programmed cell death. MAP treatment resulted in a total inhibition of ornithine decarboxylase (ODC) activity with a concomitant decrease of intracellular polyamine (mostly putrescine and spermidine) content in the breast cancer cells, whilst the spermine concentration was shown to increase. MAP proved at least 10 times more potent than the formerly studied DL-alpha-difluoromethylornithine making it an attractive candidate for clinical testing.

Gonadotropin-releasing hormone analogue conjugates with strong selective antitumor activity.

Conjugation of gonadotropin-releasing hormone (GnRH) analogues GnRH-III, MI-1544, and MI-1892 through lysyl side chains and a tetrapeptide spacer, Gly-Phe-Leu-Gly (X) to a copolymer, poly(N-vinylpyrrolidone-co-maleic acid) (P) caused increased antiproliferative activity toward MCF-7 and MDA-MB-231 breast, PC3 and LNCaP prostate, and Ishikawa endometrial cancer cell lines in culture and against tumor development by xenografts of the breast cancer cells in immunodeficient mice. MCF-7 cells treated with P-X-1544 and P-X-1892 displayed characteristic signs of apoptosis, including vacuoles in the cytoplasm, rounding up, apoptotic bodies, bleb formation, and DNA fragmentation. Conjugates, but not free peptides, inhibited cdc25 phosphatase and caused accumulation of Ishikawa and PC3 cells in the G2/M phase of the cell cycle after 24 h at lower doses and in the G1 and G2 phases after 48 h. Since P-X-peptides appear to be internalized, the increased cytotoxicity of the conjugates is attributed to protection of peptides from proteolysis, enhanced interaction of the peptides with the GnRH receptors, and/or internalization of P-X-peptide receptor complexes so that P can exert toxic effects inside, possibly by inhibiting enzymes involved in the cell cycle. The additional specificity of P-X-peptides compared with free peptides for direct antiproliferative effects on the cancer cells but not for interactions in the pituitary indicates the therapeutic potential of the conjugates.

Direct anticancer activity of gonadotropin-releasing hormone-III.

In previous studies GnRH-III, a variant of the hypothalamic neurohormone GnRH, was isolated from the brain of the sea lamprey and structurally characterized. GnRH-III is a hypothalamic neurohormone in both female and male sea lampreys. In the present work biological activities of GnRH-III in mammalian systems were examined. In superfused rat pituitary cells, GnRH-III at 1 nM to 100 nM neither induced LH-secretion nor inhibited the LH-secretion elicited by native GnRH and elicited LH release only at 1 microM. At high dose (500 microg/day) in vivo, GnRH-III behaved as a GnRH agonist, though, it was 1000-fold less active than ovurelin. The in vitro and in vivo results were in good agreement in showing that GnRH-III is only a weak agonist of the endocrine activity of GnRH. GnRH-III specifically bound to receptors on cancer cells and recognized not only the high-, but also the low-affinity binding sites. GnRH-III significantly suppressed growth of human cancer cells which have GnRH receptors. The inhibitory effect of GnRH-III on growth of cancer cells was specific and direct since the peptide did not have endocrine activity in the concentration range found to be effective in anticancer assays. GnRH-III inhibited equally the growth of ER-positive and -negative breast and TeR-positive and negative prostate cells.

Synthesis of gonadotropin-releasing hormone III analogs. Structure-antitumor activity relationships.

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.

Synthesis, conformation, biodistribution, and hormone-related in vitro antitumor activity of a gonadotropin-releasing hormone antagonist-branched polypeptide conjugate.

Since permanently high levels of GnRH analogues are necessary to exert direct and/or indirect antitumor effect on mammary tumors, much emphasis was put on the development of retarded-release devices (e.g. microcapsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) prodrugs producing prolonged release or (b) macromolecular therapeutics. In order to evaluate the feasibility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10)-GnRH (MI-1544). As a carrier, a representative of a new generation of synthetic, biodegradable branched poly[Lys(Xi-DL-Alam)] (XAK) type polypeptides with poly(L-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic polypeptide with free gamma-carboxyl groups was conjugated to MI-1544, which has only a single amino group at position 6. In this paper, we describe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544-AcEAK conjugate with a 33% degree of substitution, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone-related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the conjugate studied by in vitro assays. Data obtained with 111In- and 125I-labeled conjugates have demonstrated that in fact the body/blood survival of MI-1544 was prolonged by 1.5-3 times. The direct in vitro antitumor effect of MI-1544 was maintained or even enhanced in the MI-1544-AcEAK conjugate. Furthermore, we have shown that this conjugate was able to antagonize the effect of GnRH in vitro or to act as free MI-1544 both in short- and long-term inhibition of ovulation even after single subcutaneous injection. These data suggest that it is feasible to use a biodegradable polymeric polypeptide for development of a macromolecular therapeutic with GnRH antagonists.

Effect of gonadotropin-releasing hormone analogs and their conjugates on gonadotropin-releasing hormone receptor--positive human cancer cell lines.

The purpose of the present investigation was to develop new gonadotropin-releasing hormone (GnRH) antagonists and to increase their stability and antitumor effect by conjugation with carrier macromolecules. Antitumor effect was evaluated using clonogenic assay, cell counting for antiproliferation, and sulforhodamine B method. The presence of GnRH-binding sites in human cancer cell lines (MCF-7, MDA-MB-231, Ishikawa, LNCaP) was proved. The direct growth inhibition of tumor cell lines is achieved with relatively high analog concentrations (10(-10)- 10(-5) M). We have developed new GnRH analogs of human and chicken origin. MI-1544 (Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)GnRH and the chicken GnRH antagonist MI-1892 (Ac-D-Trp1,3, D-Cpa2, Lys5, [beta-Asp(DEA)]6, Gln8, D-Ala10)-GnRH have stronger direct antitumor properties than the agonists. The antagonists inhibited proliferation of GnRH receptor-positive human cancer cell lines by 28 to 38%. GnRH peptide analogs were coupled with macromolecules through biodegradable groups, to enhance their antitumor effects. The antagonists reduced survival of MCF-7 and MDA-MB-231 cells by 38 to 48% and 20 to 41%, respectively. They showed less activity against human endometrial and prostate cancer cells (10-20%). The copolymer (P) as polyanionic carrier molecule reached only 15 to 20% survival reduction in all cell lines. However, the copolymer GnRH antagonist conjugates P-X-1892 and P-X-1544 killed 95 to 98% of cells at doses corresponding to the GnRH analog concentration. These compounds having antitumor activity could be tried for the treatment of prostate, breast, and endometrium cancer.

In vivo studies of the new gonadotropin-releasing hormone antagonist--copolymer conjugates having antitumor activity.

The aim of the study was to test in vivo the gonadotropin-releasing hormone (GnRH) antagonists and their conjugates showing antitumor activities in vitro. The in vivo experiments with the human GnRH antagonist MI-1544 (Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)-GnRH, the chicken GnRH antagonist MI-1892 (Ac-D-Trp1,3,D-Cpa2,Lys5,/beta-Asp(DEA)/6,Gln8,D-Al a10)-GnRH, and their copolymer conjugates were carried out on MCF-7 and MDA-MB-231 human breast tumors xenografted in immunosuppressed CBA/Ca HRIJ-T6 female mice and on MXT mouse mammary tumors in BDF1 mice. The P-X-1544 and P-X-1892 conjugates were prepared by coupling the GnRH antagonists to macromolecule copolymer through biodegradable spacers. MI-1544 and its conjugate had strong, whereas MI-1892 and its conjugate had slight, castration effect in rats. All of them showed selective antitumor activity. The conjugates, given daily, inhibited both types of xenografts by 42 to 49%. Their activity was stronger in MXT mammary tumor (72 to 61%). The in vivo effect of GnRH antagonists was largely increased by coupling them to nonbiodegradable macromolecule carriers of polyanionic character. P-X-1544 and P-X-1892 GnRH antagonist-macromolecule conjugates might become important therapeutic agents for the treatment of breast cancer.

Synthesis of GnRH analogs having direct antitumor and low LH-releasing activity.

New chicken I GnRH agonists and antagonists have been synthesized and tested for their biological activities. The common feature of these analogs was that the molecules had a beta-L-aspartyl residue inserted in position 6. The agonist bound to the pituitary still had low endocrinological activity. On the other hand, it exhibited direct antitumor effect in in vitro assays. The endocrinological activity of the antagonist was low; however, it showed potent, direct antitumor activity. These observations might lead to the development of new GnRH analogs with selective antitumor effect.

Establishment and characterization of an EBNA-negative human lymphoma cell line (BHL-89).

BACKGROUND: The aim of the work was to establish human malignant lymphomas in culture, in order to study the biological characteristics and drug sensitivity of lymphomas of human lymphoid origin. MATERIALS AND METHODS: Lymph nodes of patients were explanted and kept in cultures using conventional tissue culture methods. Cytogenetic methods were used for karyotype analysis. Clonogenic assay was applied to test drug sensitivity. The tumorigenic capacity of the cells was determined by inoculating them into immunosuppressed mice. Immunological and other markers were examined with conventional techniques. RESULTS: A cell line, BHL-89, was established in culture from a patient with B-cell type non-Hodgkin's malignant lymphoma. Cells started to grow after a few days without a feeder layer in stationary suspension. The population doubling time was 48 h. The cells were hyperploid, and non-random aberrations were +1, -15, +14q+. Cloning efficiency in soft agar was found to be as high as 50-60%. The cells expressed markers characteristic of early B cells. The BHL-89 cells were Epstein-Barr nuclear antigen (EBNA) negative. They produced tumors when 10(7) cells were injected into immunosuppressed mice. The cells were sensitive to dibromodulcitol (Elobromol), an alkylating antitumor drug, and resistant to the phorbol ester TPA. CONCLUSIONS: The established EBNA-negative BHL-89 cell line has a few unique characteristics, e.g. rapid establishment without feeder cells, origin from the lymph node of an adult patient, high clonogenicity in soft agar, and resistance to TPA. The cell line is suitable for studying the nature of B lymphomas and testing compounds against lymphoproliferative disorders.

Antitumour effect of a gonadotropin-releasing-hormone antagonist (MI-1544) and its conjugate on human breast cancer cells and their xenografts.

Our gonadotropin-releasing hormone (GnRH) antagonist analogue MI-1544 ([Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10]GnRH) was developed as a potential contraceptive material, because it decreased the luteinizing hormone level without unfavourable side-effects. The antagonist was covalently bound to poly[Lys-(Ac-Glu0.96-DL-Ala3.1)] (AcEAK)-a branched polypeptide having a polylysine backbone--resulting in a MI-1544-AcEAK conjugate. According to our in vitro experiments the MI-1544 induced a 33%-35% decrease in cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines at a dose of 30 microM. The biodegradable polymeric carrier, AcEAK, alone inhibited cell proliferation by only 13%-15%, while the MI-1544-AcEAK conjugate, applied at the same dose, was capable of producing 45%-50% inhibition of cell proliferation. Our in vivo experiments using immunosuppressed mice showed that MI-1544, applied twice daily s.c., inhibited the growth of oestrogensensitive and -insensitive xenografts by 65% and 30% respectively. This effect was potentiated (70%) in both types of xenografts by the presence of the polymeric carrier in the conjugate; however, the carrier by itself did not cause tumour growth inhibition. The polymeric polypeptide carrier is supposed to increase the stability of the GnRH antagonist and to prevent the rapid excretion of the covalently bound peptide molecule. The antagonist and its conjugate may have various direct and indirect effects on breast cancer cells and, as a consequence, the new GnRH antagonist conjugates are suitable for treating an extended range of breast cancers.

Dominant expression of multidrug resistance in intraspecific murine lymphoma hybrid cells.

Cultured P388/VCR mouse lymphoma cells resistant to vincristine (VCR) and to 5-bromodeoxyuridine (BUdR) and deficient in thymidine kinase (TK-) were fused with P388/DAG cells resistant to 1.2:5,6-dianhydrogalactitol (DAG), an anticancer alkylating agent, and to 6-thioguanine (6-TG) and deficient in hypoxanthine phosphoribosyl-transferase (HPRT-). The hybrid cells expressed multidrug resistance (MDR), i.e., resistance to VCR and cross-resistance to Adriamycin (ADM) and actinomycin D (Act. D), in a dominant manner. The presence of glycoprotein p170, the MDR gene product, was detected in the hybrid cells. Resistance to DAG was also expressed dominantly, whereas cross-resistance to dibromodulcitol (DBD), a chemically related anticancer drug, was slight.

Establishment, characterization and drug sensitivity of a new anaplastic thyroid carcinoma cell line (BHT-101).

A thyroid carcinoma cell line, BHT-101, has been established in vitro from a metastatic lymph node deposit in a female patient with a non-hormone producing anaplastic, partly thyroglobulin- and thyroxine (T4)-positive papillary thyroid cancer. The cell population is heterogeneous, containing epithelial-like and fibroblast-like cells, and has a doubling time of 24 h. The cell line is polyploid with hypertetraploid predominance and the karyotype showed trisomies, tetrasomies, pentasomies as well as many marker chromosomes. The majority of the cells are negative or weakly positive for thyroglobulin and thyroxine and estrogen and progesterone receptors are present in the cells. BHT-101 cells produce tumours when injected into immunosuppressed CBA/Ca mice. The cells are sensitive to adriamycin, methotrexate and tamoxifen but not to methimazole (Favistan). The epithelial-like clone 1 and the fibroblast-like clone 3, isolated from the parental line, differed in drug sensitivity. This new cell line is suitable for studying the biology of thyroid carcinoma and for parallel in vivo and in vitro studies of drug activity against thyroid cancer.

Benzamide potentiation of the cytotoxicity of bifunctional galactitol [correction of galacticol] in resistant P388 leukemia correlates with inhibition of DNA ligase II.

Benzamide (BA) enhances the cytotoxicity of 1,2:5,6-dianhydrogalactitol (DAG) in resistant P388 leukemia cell lines but not in the sensitive parent line. To examine the reason for this difference in response, we carried out an alkaline elution assay using proteinase K to study DNA interstrand cross-linking. At early time points, equal concentrations of DAG produced the same level of interstrand cross-linking (ICL) in the resistant and sensitive P388 leukemic cells, although marked differences were observed in their cytotoxicity toward the two cell lines. In the sensitive cells, neither the amount of DNA cross-linking nor the cytotoxicity changed during the observation period (38 h) in either the presence or the absence of BA. In contrast, the elution rate of the DNA of DAG-treated resistant cells increased with time and had reached the control levels by 38 h. However, when these cells were postincubated with BA for 38 h, the elution rate of DNA was much faster than that observed for the untreated resistant cells, indicating an accumulation of DNA single-strand breaks (SSB). The SSB accumulation caused by BA was associated with an inhibition of the activity of ligase II enzyme, which was stimulated when resistant cells were treated with DAG alone. The potentiating effect of BA on the resistant cells can thus be related to the inhibiting action of BA on the DNA-rejoining enzyme, ligase II. The lack of sensitization by BA of the DAG-treated parent cell line may be attributable to the absence of DNA-SSB formation, which is necessary for ligase II activation through the stimulation of poly(ADP-ribose) synthesis.

Comparative studies on the polyamine metabolism and DFMO treatment of MCF-7 and MDA-MB-231 breast cancer cell lines and xenografts.

In order to characterize the estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and xenografts, their growth kinetic parameters and some biochemical characteristics concerning the receptor status and polyamine metabolism were determined and compared. The doubling times calculated from the growth curves showed higher proliferation rate of MDA-MB-231 cells, both in culture (21 hours) and in xenograft (9.7 days), in comparison to the MCF-7 cells which had values of 32 hours and 11.6 days, respectively. Growth-dependent changes observed in the intracellular putrescine, spermidine and spermine concentrations indicated a higher activity of polyamine metabolism in the MDA-MB-231 cells and xenograft as well. However, biosynthetic key-enzyme ornithine decarboxylase activity (ODC, EC 4.1.1.17) showed neither characteristic differences between the two types of breast cancer, nor consistent relationship with their proliferation rate. Metabolic alterations of the MCF-7 and MDA-MB-231 cell lines grown in vitro were also reflected in the polyamine composition of their culture medium. Independently of their receptor status, both types of breast cancer were responsive to difluoromethylornithine (DFMO) treatment. DFMO inhibited the ODC activity totally and depleted the cellular polyamine levels. MCF-7 cells in culture were more sensitive to the antitumoral effect of DFMO than the MDA-MB-231 line, while the rate of growth inhibition did not differ significantly in the xenografts. The present results provided further evidence on the different polyamine metabolism of ER-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells in vitro and in vivo, suggesting a correlation of hormonal modulation with polyamines as a determinant group of biological response modifiers.

Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts.

The specific binding of luteinizing hormone-releasing hormone (LH-RH) agonist in estradiol-dependent MCF-7 and estradiol-independent MDA-MB-231 human breast cancer cells has been studied using [3H]Ovurelin [(D-3H-Phe6),des-Gly10-LH-RH- ethylamide]. The results of Scatchard analyses suggest the presence of a single class of receptor sites, both in cell suspensions and membrane fractions. Evaluation of these peptide receptors appears to reflect additional characteristics of biological behaviour of these human breast cancer cells. The synthetic LH-RH agonist Ovurelin [(D-Phe6),des-Gly10-LH-RH-ethylamide] can directly interfere (25-30%) with the proliferation of MDA-MB-231 human breast cancer cells in culture. The inhibitory effect of Ovurelin in vitro was negligible in the MCF-7 cell line. In the in vivo experiments the treated immunosuppressed mice bearing either MCF-7 or MDA-MB-231 xenografts responded to the high-dose LH-RH analogue Zoladex depot and Decapeptyl depot therapy. Since the MDA-MB-231 tumour was found to be ER-negative it seems possible that the regression of this xenograft results from the direct antitumor action of the LH-RH agonist.

Genotypic and phenotypic changes of a mouse lymphoma during long-term cultivation.

NK/Ly mouse lymphoma ascites cells were explanted and established in culture. The cells grew in monolayer and showed fibroblast-like morphology. The original NK/Ly cells contained one large metacentric marker chromosome, while the in vitro growing cells had two metacentrics in the early passages. These so-called bi-armed chromosomes were growing in number, up to seven, along with the number of subcultures. No tumour "take" was observed when the cells were given into mice intraperitoneally. However, a solid tumour developed following injection of cells into the leg muscle. The histological picture of this solid tumour was anaplastic sarcoma. It is concluded that the accumulation of bi-armed chromosomes, which were formed either by anaphase bridges, or by centric fusion of telocentric chromosomes, led to a profound alteration of lymphoma resulting a phenotype of anaplastic sarcoma.

Dominance of resistance to the alkylating agent 1,2:5,6-dianhydrogalactitol in P388 mouse lymphoma hybrid cells.

Cultured P388/S mouse lymphoma cells resistant to 5-bromodeoxyuridine (BUdR) and deficient in thymidine kinase (TK-) were fused with P388/DAG cells resistant to 1,2:5,6-dianhydrogalactitol (DAG), an anticancer alkylating agent, and to 6-thioguanine (6-TG) and deficient in hypoxanthine phosphoribosyl-transferase (HPRT-). Sensitivity to DAG in the hybrid line was very close to that in the P388/DAG line, which means that resistance to DAG was inherited in a quasi-dominant manner. Hybrid cells showed cross-resistance, similar to that of the DAG-resistant line, to two other hexitols, dibromodulcitol (DBD) and disuccinyldianhydrogalactitol (DisuDAG).

Heterogeneity of the response to inducers of differentiation and to cytostatics of tumor cell populations.

The purpose of the experiments was to establish whether individual cells of a tumor cell population, or clonal lines derived from its express the differentiated phenotype, or respond heterogeneously following treatment with inducers of differentiation or with cytostatic drugs. The human cell lines used in this study were: HL-60 promyelocytic leukemia, K562 erythroleukemia, BHM-97 and A2058 melanoma, and A-1, A-2, A-4 and A-6 clones of A2058 line. Inducers of differentiation were phorbol myristate acetate (PMA), dimethylsulfoxide (DMSO) and retinoic acid (RA); cytostatics: adriamycin (ADM), 5-fluorouracil (5-FU), dacarbazine (DTIC), cis-platin (platidiam, PD) and arabinosyl cytosine (ara-C). Expression of the differentiated phenotype was shown by cell attachment (HL-60), hemoglobin production (K562), dendrit formation (A2058, BHM-97). Individual cells expressed the differentiated phenotype heterogeneously in all types of cell populations. Clone A-4 was the most, and clone A-6 the least sensitive to PMA. The drug sensitivity of the clones was different and drug-dependent. It is concluded that induction of differentiation as another approach to therapy of cancer, similar to anticancer drug therapy, also implies disadvantages due to population heterogeneity. Combinations of cytostatics with differentiation inducers might result in improved therapeutic effects.

Changes in the polyamine and nucleotide metabolism of P388 leukemia cells treated with DL-alpha-difluoromethylornithine in culture.

Influence of DL-alpha-difluoromethylornithine (DFMO) treatment on the growth kinetics, labelling index, extra- and intracellular polyamine and nucleotide concentrations was monitored in cultured P388 leukemia cells. A substantial decrease of cell proliferation was observed when the cells were continuously treated with 1-5 mM DFMO. Depletion of cellular polyamines, mostly of putrescine and spermidine, was seen with a concomitant but delayed increase of spermidine and spermine levels in the culture medium. Changes of DNA content and of labelling index of untreated and treated cells seem to indicate that DFMO arrested cells in G1/S transition. The results presented here provide additional in vitro evidence on the characteristic changes in the metabolic imbalance of ornithine in tumor cells induced by DFMO via inhibition of ornithine decarboxylase and ornithine carbamoyl transferase activities.