Difluoromethylornithine Treatment Research Articles

Median effect and long term recovery analysis of biological modifier interactions with difluoromethylornithine on the proliferation of human melanoma cells.

The ability of difluoromethylornithine (DFMO), a specific polyamine synthesis inhibitor, to interact with various biological modifiers to inhibit the colony-forming growth of human melanoma cells was determined by using the median effect principle to computer model the strength of two agent interactions. Either alpha- or gamma-IFN (interferon) in combination with DFMO resulted in a synergistic inhibition on human melanoma colony formation. For human melanoma cells which were not resistant to 13-cis RA (retinoic acid), an additive suppression on colony formation was obtained with the retinoid-DFMO combination. Dexamethasone (DEX) interacted with DFMO to yield a synergistic reduction in melanoma colony number on glucocorticoid sensitive cells and no growth enhancement with DFMO on glucocorticoid resistant melanoma lines. Human melanoma cells displayed differential long-term growth sensitivity to DFMO treatment. C8146C human melanoma cells were terminally growth-inhibited by a 96 h exposure to DFMO, in a manner which was concentration and time dependent. The proliferation of C82-7A melanoma cells was inhibited by 95% in presence of DFMO, but upon removal of DFMO the cells regained their ability to proliferate and form colonies. The simultaneous addition of DEX plus alpha-IFN plus 13-cis-RA with DFMO caused most of the human melanoma cells in these lines to become permanently growth arrested. Pre-treatment with DEX plus alpha-IFN plus 13-cis RA, but without DFMO, did not have any long term effect on the ability of melanoma cells to recover and proliferate in soft agar.(ABSTRACT TRUNCATED AT 250 WORDS)

Anchorage dependency effects on difluoromethylornithine cytotoxicity in human lung carcinoma cells

Difluoromethylornithine (DFMO), a specific, irreversible, enzyme-activated inhibitor of ornithine decarboxylase activity, the first and rate-limiting step in polyamine biosynthesis, has been shown to inhibit neoplastic cell proliferation in culture. In most cases, such inhibition is not accompanied by cell loss, with the exception of multiple cell lines of human small cell lung carcinoma (SCC), a human leukemia cell line (HL-60), and possibly the B16 melanoma cell line. The first two cell types grow as anchorage-independent suspension cultures, the HL-60 as single cells and the SCC as multicellular spheroid aggregates. Moreover, in the spectrum of human lung carcinoma cells in culture, the SCC cells respond in a cytotoxic manner to DFMO, whereas the non-small cell lung carcinoma (non-SCC) cells, which are anchorage dependent, show only growth inhibition, without actual cell loss. In the present study, we have investigated relationships between anchorage-dependent and -independent growth patterns of cells in culture and their response to DFMO treatment. Two non-SCC lung cancer cell lines, which normally grow as anchorage-dependent monolayers, show growth inhibition but no cell loss with the addition of DFMO. When these anchorage-dependent cells were forced to grow as multicellular aggregates, by coating the culture flask with Teflon, the cells developed an increased sensitivity to DFMO. They showed not only inhibition of cell proliferation but also cell death. Two SCC cell lines, which normally grow as anchorage-independent spheroids, developed adherence to the culture dishes coated with fibronectin. These cells, which show a cytotoxic response to DFMO during normal anchorage-independent growth, developed a decreased sensitivity to DFMO, showing only cell growth inhibition, but no cell death when treated during anchorage-dependent growth. Our data thus suggest that the state of anchorage dependence of lung cancer cells in culture is a critical factor in determining their response to polyamine depletion during treatment with DFMO.

Difluoromethylornithine, an effective new treatment of gambian trypanosomiasis: Results in five patients

Recent studies have shown DL-alpha-difluoromethylornithine (eflornithine), an inhibitor of polyamine biosynthesis, to be curative in various Trypanosoma species infections of laboratory animals. Five patients are described with Gambian trypanosomiasis treated in Belgium with difluoromethylornithine, using various intravenous and oral dosage schedules. Three patients had late-stage and two had early-stage disease. Difluoromethylornithine treatment was associated with clearing of parasites from blood within one to four days, a trend towards normalization of all altered biologic values associated with the disease, and marked amelioration of clinical symptoms. Side effects of difluoromethylornithine, including loose stools in three patients and both anemia, and a decrease in auditory acuity in one patient, were mild, transient, and never required interruption of drug treatment. The presence of difluoromethylornithine in cerebrospinal fluid, determined in three patients, demonstrated that difluoromethylornithine penetrates into the central nervous system. In three patients, follow-up of at least 24 months after treatment demonstrated a continued healthy state without evidence of relapse. These promising, albeit preliminary, results of difluoromethylonithine therapy, even in patients with central nervous system involvement, indicate that extended clinical trials are warranted to determine the optimal dosage regimen in patients with early- and late-stage disease.

Mucosal polyamine profile in normal and adapting (hypo and hyperplastic) intestine: effects of DFMO treatment.

The polyamines, putrescine, spermidine, and spermine, are believed to play an important role in modulating normal and adaptive intestinal mucosal growth. Polyamine synthesis is rate limited by ornithine decarboxylase (ODC) and ODC activity is specifically inhibited by -difluoromethyl ornithine (DFMO). To assess the importance of polyamines in adaptive growth we first measured mucosal polyamine profiles at different sites in the normal rat intestine and compared the results with those obtained in adaptive hypoplasia (seven days parental nutrition, TPN), in the adaptive hyperplasia of two weeks after 90% small bowel resection (SBR) or pancreatico biliary diversion (PBD). We then examined the effects of DFMO (2% in drinking water, daily from two days before surgery) on the polyamine concentrations and the adaptive response to PBD. The hyperplasia of SBR and PBD was associated with increases in all the polyamine concentrations particularly putrescine. TPN induced a modest degree of hypoplasia and little change in polyamine synthesis resulting in subnormal polyamine concentrations and significantly inhibited the mucosal adaptive response. Changes in polyamine metabolism are important in intestinal mucosal adaptation and by controlling these changes adaptive growth can be controlled.

Human lung tumor sensitivity to difluoromethylornithine as related to ornithine decarboxylase messenger RNA levels

The differential response to polyamine depletion has been studied in two types of human lung tumor cells. Small cell lung carcinoma cells die following polyamine depletion by difluoromethylornithine treatment while non-small cell lines demonstrate a typical cytostatic response. We now report that a small cell line, NCI H82, has a lower apparent capacity for polyamine biosynthesis than does a representative non-small cell, NCI H157. In subconfluent cultures, the ornithine decarboxylase activity is 25 times lower in the small cell than the non-small cell and by comparison, the polyamines in the small cell line are markedly reduced. Most significantly, levels of mRNA coding for ornithine decarboxylase are approximately 100-fold lower in the small cell than the non-small cell line, and this difference does not appear to be a result of gene rearrangement. These results suggest that differential sensitivity to polyamine depletion is related to different steady-state levels of ornithine decarboxylase mRNA.

Effects of difluoromethylornithine on proliferation, polyamine content and plating efficiency of cultured human carcinoma cells.

We investigated the effects of an irreversible inhibitor of ornithine decarboxylase, difluoromethylornithine (DFMO), on the proliferation, polyamine content, and plating efficiency of five human adenocarcinoma cell lines in vitro. DFMO inhibited the growth of all five lines when added at concentrations between 0.1 and 5.0 mM. The cell lines varied in their sensitivity to DFMO-induced cytostasis, HuTu-80 being most sensitive and HT-29 being most resistant. These differences appeared to be related to the ability of DFMO to prevent continued production of putrescine in the treated cells. Exogenous putrescine (5-50 microM) reversed the growth inhibition for all five cell lines when added 48 h after DFMO treatment. The lowest concentration of exogenous putrescine (5 microM) only restored intracellular polyamine content of DFMO-treated cells to control levels for the first 24 h after its addition. After that time, spermidine content declined once again to that observed for cells treated with DFMO alone. The higher concentrations of exogenous putrescine restored the content of all three polyamines to control levels for as much as 3 days after its addition, but did not cause a greater increase in cell growth rates than did 5 microM putrescine. These data suggest that human adenocarcinoma cell proliferation is dependent on continued polyamine biosynthesis, but that the basal content of intracellular polyamines is greatly in excess of the minimum level required to support cell growth. In the 1.0-5.0 mM concentration range, DFMO treatment for 48 h caused a slight, but statistically significant, reduction in plating efficiency for three of our cell lines, and had no significant effect on the two others.

Possible factors in the potentiation of 1-(2-chloroethy)-3- trans-4-methylcyclohexyl-1-nitrosourea cytotoxicity by α-difluoromethylornithine in 9L rat brain tumor cells

Depletion of intracellular levels of polyamines in 9L rat brain tumor cells by α -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, significantly enhanced the cytotoxicity of 1-(2-chloroethyl)-3- trans-4-methylcyclohexyl-1-nitrosourea (MeCCNU) in vitro as measured by a colony-forming efficiency assay. Administered as a single agent, DFMO was not cytotoxic to 9L cells. Treatment for 48 hr with 10, 1, 0.5 or 0.1 mM DFMO produced similar levels of polyamine depletion and similar potentiation of MeCCNU cytotoxicity. Restoration of intracellular polyamine levels by the addition of exogenous putrescine ( 1 mM) to treated cells prevented the potentiation of MeCCNU, which indicates that this phenomenon might be the result of polyamine depletion. DNA adduct formation in polyamine-depleted and control cell was studied with [14 C]-MeCCNU; no difference in monoadduct formation was found between polyamine-depleted and control cells. Experiments to determine whether polyamine depletion has an effect on enzymes involved in the repair of alkylated bases showed that the activity of O 6-methylguanine-DNA demethylase, 7-methylguanine-DNA glycosylase and 3-methyladenine-DNA glycosylace were unaffected by 48 hr of treatment with 10 mM DFMO. DFMO treatment causes a substantial increase in the intracellular content of decarboxylated S-adenosyl- l-methionine, which was reversed by addition of putrescine. The possibility that the elevation of decarboxylated S-adenosyl- l-methionine rather than the depletion of polyamines is responsible for the effects of DFMO is discussed.

Repair of oxygen-induced lung injury in adult rats. The role of ornithine decarboxylase and polyamines.

We studied the repair of lung injury in adult rats exposed to 100% oxygen for 60 h, then placed in ambient air. Lung ornithine decarboxylase (ODC) activity and polyamine (putrescine, spermidine, and spermine) content during repair were correlated with changes in lung ultrastructure. The effect of difluoromethylornithine (DFMO), a selective Irreversible ODC inhibitor, was also studied; ODC activity increased to 25-fold baseline 2 days after injury and returned to normal by 7 days. Polyamine content increased to 3-fold baseline during the first 3 days. During the same period, the number of capillary endothelial cells and the capillary surface area almost doubled, and the number of type 2 epithelial cells increased 2.5-fold. The DFMO treatment lowered ODC activities below baseline, reduced the increase in polyamine content, and also reduced the morphometric parameters described above to only 60 to 70% of the values during normal repair. It also caused a significant decrease in the number of type 1 epithelial cells during repair, suggesting that deficient replacement by differentiating type 2 epithelial cells occurred. We conclude that marked changes in lung ODC activity and polyamine content occur during the repair of oxygen-induced injury to the lung and that selective inhibition of these changes adversely affects repair.

Effect of Epidermal Polyamine Depletion on the Accumulation of Methylglyoxal Bis(guanylhydrazone) in Mouse Skin

A systemic or topical treatment of mice with alpha-difluoromethylornithine, and irreversible inhibitor of mammalian ornithine decarboxylase, produced a rapid depletion of epidermal putrescine and spermidine. When methylglyoxal bis(guanylhydrazone), another inhibitor of polyamine biosynthesis and a potent antiproliferative agent, was subsequently administered the epidermal concentration of the latter drug rose distinctly higher than without a prior difluoromethyl ornithine treatment. The combined use of these 2 antimetabolites of polyamines also profoundly depressed epidermal DNA synthesis, especially in UV-irradiated skin. A "priming" with difluoromethyl ornithine may therefore offer a means to enhance the epidermal accumulation of otherwise poorly absorbed methylglyoxal bis(guanylhydrazone).

Use of specific gravity in the measurement of cerebral edema.

Use of specific gravity in the measurement of cerebral edema.