Tumor hypoxia, p53, and prognosis in cervical cancers.

BACKGROUND: The p53 protein is involved in the regulation of initiation of apoptosis. In vitro, p53-deficient cells do not respond to hypoxia with apoptosis as do p53-normal cells, and this may lead to a relative growth advantage of cells without a functioning p53 under hypoxia. On the basis of this hypothesis, a selection of cells with a functionally inactive p53 may occur in hypoxic tumors. The development of uterine cervical carcinomas is closely associated with infections of human papilloma viruses, which may cause a degradation of the tumor suppressor gene p53, resulting in a restriction of apoptosis. Thus, cervical cancers have often a functionally inactive p53. The purpose of our clinical study was therefore to investigate the association between p53, hypoxia, and prognosis in cervical cancers in which the oxygenation status can be determined by clinical methods. MATERIAL AND METHODS: Seventy patients with locally advanced squamous cell cervical cancer Stages IIB (n = 14), IIIB (n = 49), and IVA (n = 7) were investigated in the period from 1996 through 1999. All were treated with definitive radiotherapy with curative intent by a combination of external radiotherapy plus high-dose-rate afterloading. Before therapy, tumor oxygenation was measured with a needle probe polarographically using the Eppendorf histograph. Hypoxic tumors were defined as those with pO(2) measurements below 5 mm Hg (HF5). Pretreatment biopsies were taken and analyzed immunohistologically for p53 protein expression with the DO-7 antibody. The DNA index was measured by flow cytometry. The statistical data analysis was done with SPSS 9.0 for Windows. RESULTS: The 3-year overall survival was 55% for the whole group of patients. Clinical prognostic factors in a multivariate analysis were pretreatment hemoglobin level (3-year survival 62% for patients with a pretreatment hemoglobin > or =11 g/dl vs. 27% for hemoglobin <11 g/dl, p = 0.006) and FIGO stage (Stage IIB: 65%; Stage IIIB: 60%; Stage IVA: 29%, p = 0.01). Sixty of the 70 tumors showed positive immunohistologic staining for p53 protein (transformed p53 = tp53), and 10/70 were negative (wild-type p53 = wtp53); p53 expression had no significant impact on survival (50% for tp53 vs. 79% for wtp53, p = 0.11). FIGO stage and anemia had no impact on p53 expression. Forty-nine of 70 tumors were hypoxic (HF5+), and 21 showed no hypoxia (HF5-). Hypoxic carcinomas were more frequently positive for p53 as compared to nonhypoxic tumors (27% vs. 13%, p = 0.011) and showed a trend toward a lower survival (48% vs. 70%, p = 0.07). In a further multivariate analysis, the impact of a combination of p53 expression and hypoxia on survival was examined. After adjusting for FIGO stage and pretreatment anemia, patients with wtp53 tumors had the best prognosis (3-year survival 79%) followed by tp53-HF5(-) patients (57%), and the most unfavorable prognosis was observed for tp53-HF5(+) patients (47%). The DNA index was higher in tp53 carcinomas compared to wtp53 tumors, 1.97 +/- 0.4 vs. 1.67 +/- 0.1, p = 0.05. The highest DNA index was found in hypoxic tumors with transformed p53 (2.2 +/- 3.1). CONCLUSIONS: Advanced stage and pretreatment hemoglobin level are independent prognostic factors in cervical carcinomas. The immunohistologic detection of (a functionally inactive) p53 and the presence of hypoxia had no prognostic impact, if analyzed as single parameters. However, the combination of both parameters was able to discriminate different prognostic subgroups. Moreover, hypoxic cancers were more often immunohistologically positive for tp53 protein and had a higher DNA index with the highest DNA index in tumors with both hypoxia and tp53 protein expression. These findings in summary support the theory that the tumor's microenvironment may influence the biologic behavior via hypoxia.

Radiation induced G2/M block and apoptosis in two human sarcoma cell lines with different p53 gene status.

We investigated a wild-type (wt) p53 rhabdomyosarcoma (A-204) and a mutated (mt p53) undifferentiated sarcoma cell line (US8-93) for their response to X-rays. The observation period was 0 to 96 h after irradiation. Both cell lines showed a strikingly delayed G2/M arrest and an induction of apoptosis after irradiation. Compared with the cell line A-204 (wt p53), the cell line US8-93 (mt p53) revealed a stronger G2/M arrest. In agreement with this, in terms of viability as well as the rate of apoptosis, A-204 (wt p53) showed a stronger response to irradiation than US8-93 (mt p53). We suggest that the different p53 gene status might be the cause for a different response to irradiation.

UV-induces formation of hydrogen peroxide based on the photochemistry of ketoprofen.

Ketoprofen (KP) is a potent nonsteroidal anti-inflammatory drug. However, application to the skin is problematic because the photosensitizing properties of the benzophenone moiety may cause phototoxic effects when the treated skin region is exposed to UVA light. Using capillary electrophoresis with electrochemical detection we are able to differentiate the peroxides formed during illumination of KP-containing solutions of linoleic acid. Contrary to other profens a high amount of hydrogen peroxide was found among the reaction products. For investigation of the skin damaging effect human keratinocytes were used as models. Cell viability, DNA synthesis efficiency and intracellular concentration of peroxides were determined. Viability and proliferation behavior was not altered under the influence of KP. While lower concentrations of KP (10-100 nM) led to a protection against the UVA-induced (8 J/cm2) cell proliferation damage, higher concentrations (10-100 microM) led to an amplification of the proliferation decrease. With UVB irradiation at relevant doses the effects were lower than using UVA. Furthermore, intracellular peroxide content was increased after UV irradiation and KP addition. In conclusion some efforts have to be done to avoid these side effects in the use of KP for topical or transdermal application.

Analytical methods for measuring urea in pharmaceutical formulations.

Two new methods are described for the routine determination of urea that utilize HPTLC-densitometry and colorimetry. The methods involve derivatization of urea with p-dimethylaminobenzaldehyde to a yellow-coloured compound. Validation of the methods was accomplished with respect to linearity, accuracy, reproducibility and limit of detection/quantification. Both methods were compared with an enzymatic method previously described in the literature and were found to be in close agreement. The proposed methods have the advantages of being simple, rapid and involve a single step sample preparation. Under experimental conditions HPTLC was the most sensitive method.

[DNA flow cytometry of malignant melanoma of the choroid]. / DNS-Durchflusszytometrie maligner Melanome der Chorioidea.

Thirty-five choroidal melanomas were analyzed by flow cytometry. DNA euploidy, aneuploidy and polyploidy were found in approximately 54, approximately 46, and approximately 20% of the tumors, respectively. No correlation between histological type and ploidy was found. The synthesis phase was irregular in approximately 25 and approximately 38% of the spindle and the mixed cell melanomas, respectively. The G2M peak was broadened in approximately 50% and in approximately 23% of the spindle and the mixed cell melanomas. About 20% of the malignant melanomas were highly proliferative. In 25% of the melanomas chemotherapy seemed to be useful.

[Synthesis and melanoma inhibiting properties of 4-alkylpyrocatechol-2-O-beta-D-glucopyranosiduronic acids and their esters]. / Synthese und melanomhemmende Eigenschaften der 4-Alkylbrenzcatechin-2-O-beta-D-glucopyranosiduronsäuren und ihrer Ester.

Using two sources of selection, the higher beta-glucuronidase and tyrosinase content of malignant melanomas, new transport forms are synthetized, which are toxified to quinoids, cytotoxic products by the above mentioned enzymes. These transport forms selectively inhibit the growth of melanomas. For instance, 4-methylcatechol-2-O-beta-D-glucopyranosiduronic acid (3) is synthetized by the reaction of 4-methylcatechol with tetra-O-acetyl-beta-D-glucopyranosiduronic acid methyl ester in the presence of p-toluenesulfonic acid following treatment with alkali. 3 inhibits the growth of B16 melanoma of the mouse significantly.

[Modulation of drug penetration in the skin]. / Modulation der Wirkstoffpenetration in die Haut.

Drugs administered topically on skin should act either dermally or transdermally. They have to cross the stratum corneum (SC) which is responsible for the barrier function of the skin. Most of the drugs are not able to penetrate the SC. For this kind of drugs it is necessary to search for penetration enhancers to increase drug absorption. Sometimes systemic side effects caused by drugs applied topically have to be reduced or prevented. In these cases, so called retarders or reducers are applied as penetration modulators. Penetration into and through the SC may be influenced by physical means (hydration, iontophoresis, phonophoresis, temperature) or chemical substances. Alcohols, sulphoxides, fatty acids, esters, Azone, pyrrolidones, urea and polyoles are applied as penetration enhancers. The objectives for the use of a penetration modulator are to change the solubility and diffusivity of the drug in the SC reversibly. It is necessary to distinguish between modulators which influence the lipid pathway and those which are able to modify diffusion via the polar pathway.

[Synthesis of 5-bromosalicyl-4'-chloroanilide-O-beta-D-xylopyranoside and other transport forms enzymatically activated by microbes]. / Synthese des 5-Bromsalicyl-4'-chloranilid-O-beta-D-xylopyranosids und weiterer durch Mikroben enzymatisch aktivierbarer Wirkstoff-Transportformen.

The synthesis of 5-bromosalicyl-4'-chloroanilide-O-beta-D-xylopyranoside and other glycosides of 5-bromosalicyl-4'-chloroanilide, salicylanilide, 3,5-dichlorophenol and tetrachlorohydrochinone is described. Glycosidations follow the procedures described by Latham et al. Sabalitschka and Helferich et al. These glycosides represent relatively untoxic transport-forms of drugs, which are activated to the free drug by specific enzymes of the organisms to be destroyed. This new mechanisms can help to destroy fungi and parasites in dermatology, agriculture, horticulture and cultivation of decorative plants without side effects on the host.

[Penetration kinetics of potential virustatics into the human skin]. / Penetrationskinetik eines potentiellen Virustaticums in die menschliche Haut.

Caffeic acid oxidation product (KOP), a substance effective against various human viruses, penetrates quickly from a 1% W/O-emulsion into the skin and forms a reservoir in the horny layer. In the epidermis and dermis approximately 30 min after external application KOP concentrations of 1 to 3% of the applied total quantity are achieved, which remain nearly unchanged even after longer penetration time. In addition to references for therapy derivable from this the results permit to draw conclusions about drug resorption that can be proved in animal experiments under in-vivo conditions.

[Experimental results of the optimization of external prednisolone therapy]. / Experimentelle Ergebnisse zur Optimierung der externen Prednisolontherapie.

The initial steps of any topical therapy are characterized by the degree of liberation of the agent from the ointment base and their penetration into different skin layers. A high percentage of the topically applied prednisolone does not penetrate into the skin and may be removed from the skin surface even after some time. By altering the functional structure of the horny layer and considerably increase of prednisolone liberation from ointment bases urea is a effective penetration promotor also for prednisolone. The increased prednisolone penetration into human skin from urea containing ointment correspond with the degree of blanching results by vasoconstriction test under in vivo conditions. The resulting penetration optimation of prednisolone has two possible applications in topical therapy: an increased therapeutic effect for a given prednisolone concentration and a given therapeutic effect could be obtained with a reduced prednisolone concentration.

[Skin permeation of liposomal incorporated hydrocortisone]. / Hautpermeation von liposomal inkorporiertem Hydrocortison.

With the liposomal incorporated hydrocortisone a very much improved concentration-time profile was obtained in the different layers of human skin after topical application when compared with conventional hydrocortisone in the ointment. The increased hydrocortisone penetration into human skin correspond with the degree of blanching results by vasoconstriction test. Under in vivo conditions the influence of liposomal hydrocortisone on percutaneous resorption was investigated. In guinea pigs a decreased serum concentration and urinary excretion of hydrocortisone could be demonstrated. Thus, hydrocortisone-loaded liposomes, when applied topically, act as a selective drug delivery system, it can be provide increased therapeutic efficacy and, simultaneously, decreased unwanted systemic effects. The significance of liposomal incorporated attenuative and higher potent glucocorticoids in external therapy is discussed.

[Relation of the vehicle to urea penetration in human skin]. / Vehikelabhängigkeit der Harnstoffpenetration in die menschliche Haut.

Prerequisite to any efficiency of urea in the skin is its penetration into the different skin layers. In addition to the differences in the reactivity of the skin, the ointment base is the most important factor for the intensity and the course of urea penetration. For the penetration of urea into the human skin a strong vehicle dependence can be proved. On the basis of a different penetration kinetics of urea from O/W and W/O emulsions, various efficiencies can be deduced with regard to the penetration promotion of drugs or the increase of the water-binding capacity of the horny layer. The significance of these findings for the use of urea in the external therapy is being discussed.