Stability of sulforaphane for topical formulation.

CONTEXT: Sulforaphane (SFN) is a natural compound that has been investigated as a chemopreventive agent. SFN has been shown to inhibit the activator-protein-1 (AP-1) transcription factor and may be effective for inhibition of ultraviolet (UV) induced skin carcinogenesis. This study was designed to investigate the stability of SFN as a function of pH, temperature and in various solvents and formulations. MATERIALS AND METHODS: Stability was analyzed using high-performance liquid chromatography. A potential lead formulation was identified and evaluated in vivo. RESULTS: SFN was determined to undergo apparent first-order degradation kinetics for the conditions explored. It was observed that SFN undergoes base catalyzed degradation. Buffer species and solvent type impacts stability as well. SFN was found to be very sensitive to temperature with degradation rate changing by a factor of nearly 3.1 for every 10 °C change in temperature (at pH 4.0). SFN completely degraded after 30 days in a conventional pharmaceutical cream formulation. Improved stability was observed in organic formulation components. Stability studies were conducted on two nonaqueous topical formulations: a polyethylene glycol (PEG) ointment base and an organic oleaginous base. CONCLUSION: Topically applied SFN in the PEG base formulation significantly reduced AP-1 activation after UV stimulation in the skin of a transgenic mouse model, indicating that SFN in this formulation retains efficacy in vivo.

Acetic acid, a potent stimulator of mouse epidermal macromolecular synthesis and hyperplasia but with weak tumor-promoting ability.

The effects of a single application of various dose levels of acetic acid or the weak tumor promoter, phorbol-12,13-ditetradecanoate, on the incorporation of tritiated thymidine (3H-TDR), 3H-cytidine, and 3H-leucine into DNA, RNA, and protein of mouse epidermis, respectively, were determined and compared with histologic changes in the skin. Treatment with either 500 or 833 mumoles acetic acid induced a sequential and sustained stimulation of RNA, protein, and DNA synthesis, which was followed by extensive epidermal hyperplasia similar to that reported for the strong promoter and irritant, 12-O-tetradecanoyl-phorbol-13-acetate. A dose-response relationship between the amount of acetic acid and the rate of DNA synthesis was found between the dose levels of 33 to 833 mumoles of acetic acid per application. The latter dose induced the maximum activation of 3H-TDR into DNA at 723% of control at 2 days, whereas 33 mumoles stimulated DNA synthesis earlier and peaked at 210% of control at 3 hours. Phorbol-12,13-ditetradecanoate also stimulated macromolecular synthesis in a similar sequence, though to a lesser degree. No observable inflammation and only a slight hyperplastic response were noted with phorbol-12,13-ditetradecanoate. Weekly applications of 667 mumoles of acetic acid produced a maximal tumor response of 0.73 papilloma/mouse after 32 weeks of promotion. However, a weekly dose of 677 mumoles of acetic acid was essentially inactive when given in two divided doses. When croton oil was administered twice weekly at a 0.25%-dose level, 10.2 papillomas/mouse were induced after 32 weeks of promotion. The results showed that the previously considered nonpromoting inflammatory agent, acetic acid, must be a weak promoter. However, there was no correlation between stimulated macromolecular synthesis or hyperplasia and tumor promotion when phorbol esters were compared with acetic acid.

Ionizing radiation as an initiator: effects of proliferation and promotion time on tumor incidence in mice.

Previously we have shown that a single subcarcinogenic dose of ionizing radiation followed by 60 wk of 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to the formation of squamous cell carcinoma in Sencar mice. Our previous results also indicate that TPA pretreatment prior to irradiation results in an overall increase in total tumor incidence, including both epidermal and nonepidermal tumors (D. R. Jaffe and G. T. Bowden, Radiat. Res., 106: 156-165, 1986). These studies have been expanded in CD-1 mice to further investigate the effect of the proliferative state of the skin prior to irradiation and the promotion duration after irradiation on tumor incidence. To examine the influence of the proliferative state of the skin, 17 nmol of TPA were applied to one-half of the mice 24 h prior to irradiation. The skin was irradiated using 4 MeV X-rays at a dose rate of 0.31 Gy/min. Animals received a single dose of X-rays at 0.5 or 11.25 Gy followed by twice weekly applications of TPA (8 nmol). The animals were then promoted for either 10 or 60 wk. All animals that were promoted with TPA for the same duration had a similar incidence of papillomas regardless of radiation or TPA pretreatment. Increasing the promotion duration did not significantly alter the incidence of squamous cell carcinomas at either initiation dose. At the lower initiation dose only animals that were promoted for 60 wk developed squamous cell carcinomas. TPA pretreatment at the higher dose resulted in a slight decrease in tumor incidence; however, this was not statistically significant. The incidence of basal cell carcinomas was dose dependent and appeared to be independent of TPA promotion. These data support our earlier findings that radiation can act as a weak initiator of squamous cell carcinomas and induce basal cell carcinomas in mouse skin.

DNA excision repair in ultraviolet-irradiated normal and malignantly transformed mouse epidermal cell cultures.

Pyrimidine dimer production and excision was studied in ultraviolet light (UV)-irradiated primary cultures of epidermal cells derived from perinatal mouse skin and in an in vitro malignantly transformed epidermal cell line. Dimer production increased linearly with UV dose level for both cell types. However, at any given UV dose level, there were 20% fewer thymine-containing dimers induced in the primary cultures compared to the transformed cell line. The reduced dimer yield in the primary cultures was attributed to the multilayer (three cell layers) of these cultures. The primary cultures were found to exicse no more than 10% of the original dimers in a 24-hr period, while the malignantly transformed cells excised 34%. Nonsemiconservative DNA repair synthesis was also studied as a function of dimer yields in the first 3 hr after irradiation. When the levels of repair replication in both cell types were compared at equal yields of UV-induced dimers, the malignantly transformed cells exhibited a higher level of repair than did the primary epidermal cells. There was no difference in the kinetics of repair replication between the two cell types at a UV dose level of 10 J/sq m over the first 6 hr after irradiation.

Roles of Akt and glycogen synthase kinase 3beta in the ultraviolet B induction of cyclooxygenase-2 transcription in human keratinocytes.

Ultraviolet B (UVB)-induced cyclooxygenase-2 (COX-2) expression plays an important role in UVB tumor promotion. We examined whether Akt and glycogen synthase kinase 3beta (GSK-3beta), components of the phosphatidylinositol 3'-kinase pathway, are involved in UVB induction of COX-2 transcription. UVB caused Akt phosphorylation at both Thr-308 and Ser-473 that was inhibited by LY294002, a phosphatidylinositol 3'-kinase inhibitor. LY294002 also decreased the expression of endogenous COX-2 protein and a luciferase construct driven by COX-2 promoter. Similarly, UVB caused phosphorylation of GSK-3beta (Ser-9) and presumably inactivation of GSK-3beta. Inhibition of GSK-3beta by lithium induced endogenous COX-2 protein expression and COX-2 promoter activity. Finally, overexpression of a dominant-negative Akt mutant or wild-type GSK-3beta suppressed UVB-mediated induction of COX-2 promoter. These studies suggest that inactivation of GSK-3beta through activation of Akt plays an important role in the UVB induction of COX-2 transcription.

Effect of retinoic acid on the late-stage promotion of transformation in JB6 mouse epidermal cells in culture.

beta-All-trans-retinoic acid (RA) inhibited the anchorage-independent growth of JB6 cells induced by either mezerein or alpha-epidermal growth factor (alpha-EGF) (a purified fraction of epidermal growth factor). The inhibition was dose dependent for alpha-EGF as well as for RA. Mezerein-induced growth in soft agar was inhibited to a greater extent by RA than was alpha-EGF-induced growth in soft agar, at similar colony yields. The extent of inhibition of anchorage-dependent growth induced by RA was similar for nontransformed JB6 cells and for alpha-EGF-transformed cells, so that transformation was shown not to influence the sensitivity of cells to retinoid inhibition of anchorage-dependent growth. RA was as effective at inhibiting anchorage-independent growth when it was applied after promoter-induced transformation as when it was applied during promoter-induced transformation. Therefore, the antiproliferative effect of RA, without an additional antitransformation effect, was sufficient to account for the reduced colony yield. These results suggest that the antipromoting action of retinoids in JB6 cells may occur by limiting proliferation, the regulation of which may be coupled with the state of differentiation of cells.

Influence of the duration of topical 13-cis-retinoic acid treatment on inhibition of mouse skin tumor promotion.

The effect of the time and duration of retinoid treatment on the inhibition of Stage II tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied in CD-1 mice. All mice were initiated with 400 nmol of benzo(a)pyrene and received Stage I tumor promotion (3.2 nmol of TPA twice weekly for 2 wk). Animals were then randomized into groups which received 13-cis-retinoic acid during early, middle, or late Stage II promotion. 13-cis-Retinoic acid pretreatments starting on Day 1, Wk 8, or Wk 23 of Stage II promotion resulted in 47, 28, or 19% inhibition, respectively, of TPA-induced tumor formation. One-half of the mice receiving 13-cis-retinoic acid at Day 1 or Wk 8 were removed from the retinoid treatments at Wk 23, the time of cessation of TPA promotion. The inhibition of tumor formation remained constant during the 15-wk observation period after cessation of retinoid treatment, suggesting that retinoid inhibition of mouse skin tumor promotion is stable in the absence of further promotion and preceded the step of irreversible conversion of promoter dependence to promoter independence.

Inhibition of ionizing radiation recovery processes in polyamine-depleted Chinese hamster cells.

Polyamines are involved in many cellular processes, including DNA structure and function. Since DNA, or some DNA-containing structure, is known to be the target for cell killing induced by ionizing radiation and a number of chemotherapeutic agents, we investigated the effects of polyamine depletion on cytotoxic responses of Chinese hamster cells to X-irradiation. Colony forming ability after single, acute radiation exposures of cells growing under oxic conditions was minimally affected by endogenous putrescine and spermidine depletion, achieved after treatment with alpha-difluoromethylornithine. Survival of cells rendered hypoxic and then irradiated was unaffected by alpha-difluoromethylornithine treatment. However, cellular recovery processes were nearly completely suppressed in polyamine-depleted cells, including sublethal damage recovery, as evidenced by split-dose irradiations in log phase cultures, and potentially lethal damage recovery, observed when growth-inhibited cultures were allowed time to repair radiation damage prior to being plated for colony formation. Both these recovery processes were restored by exogenous putrescine treatment. Reaccumulation of intracellular spermidine content closely correlated with restoration of potentially lethal damage recovery. Depletion of putrescine and spermidine pools had little effect on either single or double strand DNA break production or rejoining. These data demonstrate that both sublethal and potentially lethal damage recovery are polyamine-dependent processes in Chinese hamster cells, and imply that the mechanisms by which hamster cells recovery from these types of radiation damage are unrelated to their ability to rejoin DNA strand breaks, at least during the first hour after irradiation. Finally, these results suggest that the depletion of tumor polyamine content may be an effective method of enhancing the sensitivity of human tumors to fractionated radiotherapy.

Effects of dietary retinyl palmitate or 13-cis-retinoic acid on the promotion of tumors in mouse skin.

The present study was designed to determine the effects of dietary 13-cis-retinoic acid and retinyl palmitate on mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Female CD-1 mice were initiated with 150 nmol of 7,12-dimethylbenz(a)anthracene and promoted twice weekly with 8 nmol of TPA. Diets supplemented with retinyl palmitate to yield 60,000 or 200,000 IU or 700,000 for 5 wk followed by 350,000 IU per kg of diet (700,000/350,000) fed to mice during tumor promotion resulted in 9%, 37%, and 65% inhibition of the papilloma yield, respectively, at 21 wk of promotion. Although topical applications of 13-cis-retinoic acid have been almost as effective as retinoic acid in preventing the appearance of mouse skin tumors, dietary 13-cis-retinoic acid at 200,000 or 700,000 IU per kg of diet resulted in no reduction in papilloma yield but did result in a dose-dependent decrease in the tumor burden (weight of tumors per mouse). Therefore, dietary retinyl palmitate yielded a dose-dependent inhibition of the number and weight of tumors promoted by TPA, whereas dietary 13-cis-retinoic acid resulted in a decrease in weight but not in number of tumors promoted by TPA.

Excision of pyrimidine dimers from epidermal DNA and nonsemiconservative epidermal DNA synthesis following ultraviolet irradiation of mouse skin.

Pyrimidine dimer production and excision in epidermal DNA were studied at five different dose levels of ultraviolet light in the skin of intact mice. Dimer production increased with dose up to 50,400 ergs/sq mm. Approximately 30% of the thymine-containing dimers were excised by 24 hr after irradiation at three lower dose levels of ultraviolet light. Nonsemiconservative DNA replication in ultraviolet-irradiated mouse skin was shown to continue for at least 18 hr. The rate of nonsemiconservative replication decreased with time, but did so slowly. The initial rates of nonsemiconservative replication increased uith ultraviolet light dose level up to about 4,200 ergs/sq mm, after which the initial rates were decreased. Semiconservative epidermal DNA synthesis was shown to be inhibited by hydroxyurea, but hydroxyurea had no effect on ultraviolet light-induced nonsemiconservative DNA replication. The observed pyrimidine dimer excision and nonsemiconservative DNA replication suggest that in the intact mouse the cells of the epidermis are capable of DNA excision repair after ultraviolet irradiation of mouse skin.

Characterization of hepatic DNA damage induced in rats by the pyrrolizidine alkaloid monocrotaline.

Hepatic DNA damage induced by the pyrrolizidine alkaloid monocrotaline was evaluated following i.p. administration to adult male Sprague-Dawley rats. Animals were treated with various doses ranging upward from 5 mg/kg, and hepatic nuclei were isolated 4 hr later. Hepatic nuclei were used as the DNA source in all experiments. DNA damage was characterized by the alkaline elution technique. A mixture of DNA-DNA interstrand cross-links and DNA-protein cross-links was induced. Following an injection of monocrotaline, 30 mg/kg i.p., DNA-DNA interstrand cross-linking reached a maximum within 12 hr or less and thereafter decreased over a protracted period of time. By 96 hr postadministration, the calculated cross-linking factor was no longer statistically different from zero. No evidence for the induction of DNA single-strand breaks was observed, although the presence of small numbers of DNA single-strand breaks could have been masked by the overwhelming predominance of DNA cross-links. These DNA cross-links may be related to the hepatocarcinogenic, hepatotoxic, and/or antimitotic effects of monocrotaline.

Molecular pharmacology of the anthracycline drug 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1 H-imidazol-2-yl)hydrazone] dihydrochloride (Cl 216,942).

9,10-Anthracenedicarboxyaldehyde bis[(4,5-dihydro-1H-imidazol-2yl)hydrazone] dihydrochloride (Cl 216,942) is a new anthracene bishydrazone derivative which has shown antitumor activity in Phase I trials against both hematological cancers and solid tumors. The effects of Cl 216,942 on L1210 mouse leukemia cells were studied with the nucleoid sedimentation and alkaline elution assays. Evidence for Cl 216,942 intercalation into cellular DNA was obtained in exponentially growing cells by comparing the L1210 nucleoid sedimentation behavior in neutral sucrose gradients of ethidium bromide with nucleoids from Cl 216,942-treated cells. A 1-hr treatment of exponentially growing L1210 cells with Cl 216,942 induced both protein-associated DNA single-strand breaks and DNA-protein crosslinks as detected by the alkaline elution assay. The DNA strand break and DNA-protein cross-link frequencies were found to be within a factor of 2 of each other over a range of Cl 216,942 concentrations. The dose response for the induction of DNA damage showed a linear decrease up to 10 micrograms/ml, but this was followed by a decrease in damage at dose levels greater than 10 micrograms/ml. The biphasic dose response could not be explained by changes in the cellular uptake of Cl 216,942. The kinetics of Cl 216,942 induction of DNA damage after a 1-hr treatment showed that at the dose which gave maximum damage the degree of damage (10 micrograms/ml) decreased with further incubation, but at a higher dose (20 micrograms/ml) DNA damage increased with postincubation at 37 degrees. The cytotoxicity produced by Cl 216,942 at a given frequency of protein-associated strand breaks was low. Cl 216,942 thus appeared to belong to a low-toxicity group of DNA intercalators.

Tumor promoter-induced inhibition of epidermal growth factor binding to cultured mouse primary epidermal cells.

The effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and other diterpene derivatives on the binding of epidermal growth factor (EGF) to primary cultures of mouse epidermal cells was studied. 125I-EGF was used to study the specific binding of the growth factor to monolayer cultures of mouse epidermal cells grown under low-calcium culture conditions (0.06 mM Ca2+). Under these growth conditions, nonspecific binding did not exceed 10%. Initially, TPA decreased the binding of 125I-EGF to cells. However, when cells were incubated continuously in TPA plus EGF (0.25 ng/ml) for 19 hr, there was more EGF bound to the TPA-treated cells than to control cells. This phenomenon was not observed at high (5 ng/mg) EGF concentrations. Scatchard analysis of specific 125I-EGF binding at 4 degrees after a 1-hr pretreatment of the cells with TPA at 37 degrees converted a curvilinear plot to a linear plot. TPA induced a 25% decrease in the number of receptors per cell and eliminated binding of EGF to a class of high-affinity receptors. Preincubation of cells in TPA at 37 degrees for up to 13 hr followed by Scatchard analysis at 4 degrees showed that the curvilinear plot was restored and that the effects of TPA were partially reversible. TPA did not alter the rate at which bound EGF was degraded. However, at low EGF concentrations, TPA reduced the amount of EGF that was metabolized. The greater amount of EGF bound to TPA-treated cells over controls after long-term incubation was due to the presence of larger amounts of whole EGF in the media of TPA-treated cells at a time when the cells have regained their ability to bind EGF. A series of diterpene derivatives of different abilities to act as tumor promoters and hyperplasia-inducing agents were tested for their ability to influence EGF binding. The abilities of members of this series to decrease EGF binding and prevent degradation of EGF correlated more with their potentials to induce hyperplasia than with their tumor-promoting potentials. The ability of these diterpene derivatives to induced DNA synthesis with EGF synergistically may depend on the transient sparing of the EGF from degradation and subsequent binding of the spared EGF.

Oncogene activation and tumor suppressor gene inactivation during multistage mouse skin carcinogenesis.

The mouse skin multistage model of carcinogenesis is an ideal system in which to study questions related to the timing of oncogene activation and inactivation of tumor suppressor genes. A number of laboratories have shown that an early event associated with chemical initiation of mouse skin tumors involves activation of the Harvey-ras oncogene. To approach the question of timing of loss of tumor suppressor genes in skin carcinogenesis, we have utilized a model system developed by Kulesz-Martin in which cloned mouse keratinocytes were initiated with DMBA and variant clones with benign or malignant phenotypes were developed. We have generated somatic cell hybrids between the parental clone and the variants to study the potential loss of tumor suppressor activity during the progression of cells from the initiated to benign and to the malignant phenotypes. Somatic cell hybrids generated between the parental, normal cell strain (i.e., 291) and a malignant cell variant (i.e., 05), that produces moderately differentiated squamous cell carcinomas (SCCs), failed to produce tumors indicating tumor suppressor activity in the 291 cells. The 291 cells and a benign papilloma producing variant (i.e., 09) were able to partially suppress in hybrids the tumorigenicity of another malignant cell line (i.e., 03) which produces poorly-differentiated SCCs. Suppression of 03 tumorigenicity by the benign tumor cell, 09, was less than that seen with the normal cell, 291. These results indicated two potentially different suppressor activities were inactivated during progression of normal 291 to malignant 03 cells. We have also obtained evidence that constitutive AP-1 activity plays a role in the maintenance of the malignant phenotype of SCC cell lines. Two different SCC cell lines, 308 10Gy5 and PDV, demonstrate constitutive AP-1 activity. To examine the role of this activity in malignant progression, we stably expressed a transactivation deletion mutant of the human c-jun gene in these cell lines. Expression of this mutant c-jun protein blocked transcriptional transactivation of AP-1 responsive reporter CAT constructs driven by jun, human collagenase, and the mouse stromelysin promoters. These malignant cells were not only inhibited in their AP-1 transactivation response, but also in their ability to form SCCs upon s.c. injection into athymic nude mice. These results support the idea that inhibition of AP-1-mediated transcriptional transactivation is in some cases sufficient to suppress the tumorigenic phenotype of malignant mouse epidermal cells.

Interactions of mitomycin C with mammalian DNA detected by alkaline elution.

The antitumor antibiotic mitomycin C (MMC) was studied in vitro using L1210 leukemia and 8226 human myeloma cells. Cytotoxicity was evaluated by colony formation in soft agar, and DNA damage was analyzed using alkaline elution filter assays. The purposes of these studies were: (a) to characterize the time course of MMC-DNA damage; (b) to characterize the type of DNA damage [DNA-DNA interstrand cross-links (ISC), DNA-protein cross-links (DPC), single and double strand breaks (SSBs, DSBs)]; and (c) to correlate this damage with cytotoxicity in vitro. Colony-forming assays showed the D0 value for 1 h MMC to be 15.0 microM for L1210 cells and 17 microM for 8226 cells. Alkaline elution studies showed that dose-dependent ISCs and DPCs formed rapidly following MMC exposure. Removal of cross-links was delayed, with only 50% repaired 32 h after exposure. There was a good correlation between ISCs and cytotoxicity in dose-response studies in each cell line. ISCs appeared to comprise most of the MMC-DNA lesions in both cell lines. No DNA SSBs or DSBs were observed following MMC exposure. Nuclei isolated from both cell lines and exposed to MMC produced less MMC alkylation than whole cells but, again, no strand breaks were evident. These results demonstrate that MMC is principally an alkylating agent when used at pharmacological (cytotoxic) concentrations in vitro. The lack of evidence for DNA strand breaks discounts a significant role for putative quinone-generated oxygen free radicals in the production of MMC cytotoxicity.

Inhibitory effects of perillyl alcohol on UVB-induced murine skin cancer and AP-1 transactivation.

The monoterpene perillyl alcohol (POH) has proven efficacious against the formation and progression of a variety of cancers. In this study, we tested the ability of POH to inhibit photocarcinogenesis in a nonmelanoma model of mouse skin carcinogenesis and its ability to inhibit UVB-induced activator protein 1 (AP-1) transactivation in mouse skin and human keratinocytes. POH (10 mM) was applied topically to the ears and shaved dorsal surface of groups of 35 BALB/c mice throughout the experiment, during and after UVB treatment. Topical POH significantly inhibited tumor incidence and multiplicity, average tumor size, and the average tumor burden/mouse without any apparent toxicity. POH inhibited UVB-induced AP-1 transactivation in both cultured human keratinocytes and transgenic mice that stably express a luciferase reporter driven by AP-1 elements. The results suggest that POH might be used for chemoprevention of human skin cancer, and that inhibition of AP-1 activity is functionally related to inhibition of skin carcinogenesis.

Transient alterations in the expression of protease and extracellular matrix genes during metastatic lung colonization by H-ras-transformed 10T1/2 fibroblasts.

It has been proposed that tumor progression is a selective process and that only a minority of tumor cells survive this selection because they possess the phenotypic traits necessary for metastasis and organ colonization. Both proteases and extracellular matrix proteins have been implicated in invasion and metastasis formation. To examine the nature of the selection process, we transformed 10T1/2 fibroblasts with T24 H-ras and the neoR gene and selected a clonal line expressing the mutant ras gene. After i.v. injection of this line into syngeneic C3H/HeN mice, tumor cells were recovered from lungs by enzymatic treatment and selective outgrowth in G418. Less than one of 10(3) cells survived in the lung 30 min after inoculation, and these exhibited a unique phenotype. This was characterized by a propensity to lodge in the lung on reinjection; markedly enhanced mRNA levels of procollagen alpha 2(I), procollagen alpha 1(III), and fibronectin; and decreased levels of laminin, major excreted protein (procathepsin L), transin, and H-ras. Between 1 and 9 days after tumor injection, the phenotype of the cells surviving in the lung changed dramatically and exhibited a pattern of gene expression with increased protease and low matrix protein mRNA levels. This coincided with a 26-fold increase in the ability to colonize lungs on i.v. injection. Both the phenotype characterized by its propensity to arrest in the lung and that showing enhanced metastatic ability were unstable on prolonged in vitro culture. We hypothesize that two selection events have occurred. The first is for lung arrest and implantation of variants of the injected tumor with high matrix protein and low protease levels. A second selection then occurs for tumor cells that carry a favorable phenotype for invasion and proliferation which is associated with low matrix protein and high protease gene expression. These two phenotypes are represented within a clonal population of recently transformed tumor cells.

Comparative molecular pharmacology in leukemic L1210 cells of the anthracene anticancer drugs mitoxantrone and bisantrene.

1,4-Dihydroxy-5,8-bis(2-[(2-hydroxyethyl)aminoethyl]amino)-9, 10-anthracenedione (mitoxantrone) and 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2y)hydrazone] dihydrochloride (bisantrene) were evaluated for their abilities to cause cytotoxicity and interact with cellular DNA using leukemic L1210 cells. On a molar basis mitoxantrone has been found to be 7-fold more toxic than bisantrene. Using a nucleoid sedimentation technique, bisantrene caused changes in DNA supercoiling which were characteristic of an intercalating drug, but mitoxantrone did not induce these changes. Both drugs were found to interact with cellular DNA with tight but noncovalent binding. Both drugs also induced protein-associated double- and single-strand DNA breaks, but with mitoxantrone only some of the DNA single-strand breaks were protein associated, whereas with bisantrene all the DNA single-strand breaks were protein associated. The cytotoxicity produced by bisantrene at a given frequency of protein-associated DNA strand breaks was low. However, with mitoxantrone at an equivalent DNA strand break frequency, the cytotoxicity was high. Treatment of isolated L1210 nuclei with either drug did not result in DNA single-strand breaks. It can be concluded that bisantrene binds DNA in whole cells by an intercalative mechanism, whereas mitoxantrone binds DNA by a nonintercalative, electrostatic interaction and induces non-protein-associated DNA strand breaks.

Expression of the metalloproteinase matrilysin in DU-145 cells increases their invasive potential in severe combined immunodeficient mice.

Human prostate cancer displays a high degree of variability in its rate of spread, which could be due largely to differences in the invasive potential of the tumor cells. The degradation of the basal lamina and stromal extracellular matrix is mediated in part by the secretion of matrix metalloproteinases (MMPs). Matrilysin (PUMP-1, MMP-7) and gelatinase A (M(r) 72,000 type IV collagenase, MMP-2) have been shown to be overexpressed in prostate carcinoma. We have expressed the single MMP matrilysin in the tumorigenic but nonmetastatic human prostate tumor cell line DU-145 to determine if matrilysin has a functional role in prostate tumor cell invasion. DU-145 cells expressing matrilysin were significantly more invasive than vector-only transfected cell lines as assayed by a severe combined immunodeficient mouse model of tumor cell invasion. Vector-only transfected DU-145 cells injected i.p. into severe combined immunodeficient mice invaded the diaphragm in only 1 of 9 mice (11%), whereas matrilysin-transfected DU-145 cells invaded the diaphragm in 12 of 18 mice (66%). The difference between the controls and matrilysin-transfected cells was statistically significant (P < 0.006). These results suggest a functional role for matrilysin in the initial invasion of prostate cancer through the epithelial basal lamina and into the surrounding stroma.

Comparison of two-stage epidermal carcinogenesis initiated by 7,12-dimethylbenz(a)anthracene or N-methyl-N'-nitro-N-nitrosoguanidine in newborn and adult SENCAR and BALB/c mice.

In order to define factors which determine susceptibility to chemical carcinogenesis, mice sensitive (SENCAR) and resistant (BALB/c) to epidermal carcinogenesis were studied under several treatment conditions for sensitivity to initiation by 7,12-dimethylbenz(a)anthracene or N-methyl-N'-nitro-N-nitrosoguanidine and promotion by 12-O-tetradecanoylphorbol-13-acetate. In newborns of both strains, topical application of initiator was much less effective than in adults. However, initiation by i.p. injection of 7,12-dimethylbenz(a)anthracene is at least as effective in newborns as in adults, which may indicate that topically applied carcinogen is not delivered effectively to target cells in newborns. Thus, newborn epidermis can respond to 7,12-dimethylbenz(a)anthracene as well as adult epidermis when the initiator is appropriately administered. SENCAR mice are much more sensitive than are BALB/c mice to both initiators, which suggests that enhanced metabolic activation of hydrocarbon carcinogens by SENCAR mice is unlikely to account for their sensitivity. Newborn male SENCAR's developed approximately 50% more papillomas than did females in all groups. BALB/c newborn mice developed so few tumors that a meaningful comparison of sensitivity of males and females could not be made. Thus, the increased sensitivity of SENCAR's was apparent regardless of route of administration of initiator or the age or sex of the mice. SENCAR mice also developed a significant number of papillomas and squamous cell carcinomas with 12-O-tetradecanoylphorbol-13-acetate promotion in the absence of an exogenous initiator. Therefore, the skin of SENCAR mice may contain an initiated population of cells capable of responding to tumor promoters.