Klaus Goerttler

Keratin polypeptide composition as a biochemical tool for the discrimination of benign and malignant epithelial lesions in man

SummaryAn investigation was undertaken of the keratin polypeptides of various benign and malignant tumors of the human skin and vaginal epithelium by one and two dimensional gel electrophoresis. The keratin patterns of benign tumors were found to be similar to the patterns of normal epithelium or stratum corneum. The relative proportion of stratum-corneum associated keratin polypeptides to those polypeptides characteristic for the living layers corresponds to morphological features (e.g., hyperkeratosis, acanthosis). In contrast to benign tumors, epithelial carcinomas totally lack the group of high-molecular-weight keratins. This finding may be helpful in the diagnostic discrimination between benign and malignant epithelial lesions.

Diaplacental carcinogenesis: Tumor localization and tumor incidence in NMRI mice after diaplacental initiation with DMBA and urethane and postnatal promotion with the phorbol ester TPA in a modified 2-stage Berenblum/Mottram experiment

Diaplacental initiation with the carcinogens DMBA or urethane followed by repeated topical treatment of mice of the F1 generation with the tumor promoter TPA leads to the formation of benign and malignant tumors on the skin of the back as well as in other tissues and organs. The tumor yield in this modified 2-stage Berenblum/Mottram experiment considerably exceeds the number of spontaneously formed tumors and of tumors produced by initiation alone. Further differences can be demonstrated in the malignancy rate, the formation of multiple tumors in various organs, additional non-neoplastic alterations and in a reduction of the lifetime of the animals. The effect of the tumor promoter TPA is not restricted to carcinogenesis in the back skin. Obviously, TPA is able to activate inititated tumor cells in internal organs to form tumors. This, in turn, implies the absorption of the substance via the blood vessels and its distribution throughout the body. The preferential occurrence of tumors in the genital tract of female mice (carcinomas and sarcomas of the vaginal wall, granulosa cell tumors of the ovaries) points to a possible hormonal involvement; in this context, relevance to prenatally induced tumors in human pathology is discussed. The results emphasize the important role of prenatal carcinogenesis and indicate the increased risk to man by either prenatal initiation or postnatal promotion.

Two-stage skin carcinogenesis by systemic initiation of pregnant mice with 7,12-dimethylbenz(a)anthracene during gestation days 6–20 and postnatal promotion of the F1-generation with the phorbol ester 12-tetradecanoylphorbol-13-acetate

SummaryThe DMBA-TPA-mediated two-stage skin carcinogenesis experiment was modified in that pregnant mice were systemically treated once during pregnancy with the carcinogen. Intragastric application times were fetal days 6, 8, and 10–20. A control group of pregnant mice received repeated doses (from days 8–20) of sesame oil, which was used as a solvent for DMBA.At the age of 12 weeks, the offspring of the control group were divided into two groups, one of which was left completely untreated, the other received TPA applications over 26 weeks.The 12-week old F 1-progeny of each transplacentally initiated group was also divided into subgroups, which either received no further treatment (subgroups A) or were promoted with TPA (subgroups B). Neither the F 1-animals of the two control groups nor that of the transplacentally initiated but postnatally not promoted subgroups 6A–20A developed skin tumors. The same holds true for the TPA-promoted offsprings of mother animals which had received DMBA at days 6 and 8 of gestation.Skin tumor development after TPA promotion was first observed in animals of subgroup 10B. Thereafter, tumor rates and tumor yields increased and latency periods decreased progressively in the B-subgroups with the postponenment of initiation to later fetal periods. Day 19 of prenatal development proved to be the most sensitive period to transplacental initiation, whereas initiation at day 20 led to a significant decrease in tumor rate and yield.The capability to initiate skin tumors and the extent of initiation can be correlated to both the organogenesis of the epidermis and its proliferative rate in utero.

Positive two-stage carcinogenesis in female sprague-dawley rats using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and 12-o-tetradecanoylphorbol-13-acetate (TPA) as promotor

In contrast to a previous report by Shubik, the validity of the 2-stage skin carcinogenesis experiment was demonstrated in the rat. The modified experiment was carried out in female Sprague-Dawley rats using intragastrically administered DMBA as a carcinogen and the topically applied phorbol ester TPA as a promoter. Seven groups of animals were used. Two groups were treated with TPA only, two groups were initiated only with DMBA, two further groups were both initiated and promoted, and one group served as a control. Each of the initiated/promoted groups or only initiated or promoted groups contained one sub-group in which the animals had been bilaterally ovarectomized prior to the experiment. Hyperplasia of the dorsal epidermis occurred only in the promoted and in the initiated/promoted groups. Tumors of the back skin were observed exclusively after initiation/promotion. Ovarectomy — leading to a prolonged survival time of the animals — seems to be crucial for the manifestation of malignant skin tumors. Initiation/promotion also gives rise to tumors of the forestomach, the small intestine, the liver and the colon. Tumors in other organs (especially in the mammary gland and the Zymbal gland) were also be observed after initiation alone.

Protein synthesis during fetal development of mouse epidermis: II. Biosynthesis of histidine-rich and cystine-rich proteinsin vitro andin vivo

The biosynthesis of specific epidermal proteins at different stages in the development of fetal mouse epidermis has been investigated. Skin pieces from 16- to 17-day or 18- to 19-day fetal mice were incubatedin vitro with the individual amino acids [3H]histidine or [35S]cystine, or with a mixture of14C-labelled amino acids. Labelled proteins were extracted, then separated by SDS-polyacrylamide gel electrophoresis, and the newly synthesised proteins were identified by gel fluorography. The results demonstrated that several histidine-rich proteins (HRP) in the MW range 70,000–120,000 are very rapidly synthesisedin vitro. At least one of these proteins was actively synthesised in epidermis from 16- to 17-day mice, i.e., at a stage which precedes the appearance of histidine-rich keratohyalin granules. Noin vitro incorporation was observed at any stage into the major HRP constituent of mouse epidermis (MW 27,000; Balmainet al., 1977), although this protein was strongly labelledin vivo. Incorporation of [35S]cystinein vitro andin vivo indicated that a protein(s) of MW 66,000 is very rich in this amino acid.

Spontaneous tumors and lifespan of female NMRI mice of the outbred stock sut: NMRT during a lifetime study

SummaryA group of 150 female NMRI mice of the outbred stock Sut: NMRT was kept until they died naturally, at which time they were necropsied and examined histologically for spontaneous tumors. The natural life expectancy (median) was 782 days. Life expectancy was markedly reduced by mammary and pulmonary adenocarcinoma, and by tumors of the hypophysis. The spontaneous tumor rate was 58%. That is to say, 87 of the 150 mice had spontaneous tumors: 57 animals each had one tumor, 20 animals each had two tumors, and 10 animals each had three tumors. The organs most commonly affected by tumors were those of the lymphoreticular and haematopoietic systems, followed by the respiratory tract in second place and the breast in third place. Data reported in the literature generally show the same organ distribution, but the total tumor rate given is generally somewhat lower as the animals are seldom left alive until they die naturally (spontaneously).

Long term tumorigenicity of a single application of indomethacin or Amuno® in adolescent and in adult male Sprague Dawley rats

200 adolescent (Group I) and 200 adult male Sprague Dawley rats (Group II) were divided into 4 subgroups of 50 animals each. Animals were treated on the 29th (Group I) or 98th day of life (Group II) either with acetone or Amuno carrier in acetone or Amuno on acetone (2.5 mg indomethacin/100 g animal weight in acetone) or with the pure substance indomethacin 2.5 mg/100 g of animal weight in acetone, giving a single application on the shaved dorsal skin. Subsequently the animals remained under observation until their deaths, followed by autopsy and histopathologic examination of several organs. The rats treated with Amuno of indomethacin in the adolescent stage showed lower body weights and a shorter total survival time. The adolescent animals treated with Amuno or indomethacin showed a significantly higher rate of interstitial testicular tumors of the Leydig tumor type, adenomas and adenocarcinomas of the small and large intestine as well as hepatocellular tumors. The total number of neoplasias was higher in the animals treated with Amuno or indomethacin compared to those treated with acetone or carrier with acetone. Application of Amuno or indomethacin also resulted in a higher rate of hyperplasias (sole of foot, lymph nodes, prostate). The impairment of the synthesis of prostaglandins caused by indomethacin apparently results in starting complex pathomechanisms which have effects until the late death of the animals. Adolescent animals were affected more frequently by the application of indomethacin as were already adult animals.

Epidermal intercellular relationships during carcinogenesis and cocarcinogenesis as revealed by scanning electron microscopy.

SummaryInvestigations with the scanning electron microscope were carried out on the skin of 80 NMRI mice after treating them with small doses of the carcinogenic substance DMBA and the cocarcinogenic agent TPA, respectively. The results were correlated with histologic, transmission electron microscopic and autoradiographic observations. The epidermis of TPA-treated animals was markedly hyperplastic with an orderly arrangment of cell layers. Autoradiographically only the basal cells were heavily labelled. With the scanning and transmission electron microscope a reduced number of intercellular connections and dilatation of the intercellular spaces could be detected. After treatment with DMBA the epidermis was only moderately hyperplastic but severely dysplastic with3H-thymidine-labelled cells in the upper layers. The most characteristic findings were the loss of the intercellular connections, especially the lateral ones, and a pronounced dilatation of the intercellular spaces. The results obtained with the scanning electron microscope were quantified using morphometrical methods.

Effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and its nonpromoting analogue 4-O-methyl-TPA on dorsal dermal melanocytes of the Syrian golden hamster (Mesocricetus auratus)

SummaryThe effect of the tumor promoter TPA and its inactive structural analogue 4-O-methyl-TPA on the induction of dorsal skin melanosis in the normal Syrian golden hamster and on the promotion of melanomas in DMBA-initiated animals was investigated. Both phenomena were observed in TPA-treated hamsters but could not be detected after exposure of animals to 4-O-methyl-TPA. In contrast to results obtained with a variety of other laboratory animals, neither TPA nor 4-O-methyl-TPA were able to induce epidermal hyperplasia of hamster dorsal skin.

Electrolyte and glucose metabolism in VX-2 carcinoma of the rabbit

Biochemical and other parameters in VX-2 carcinoma in rabbits were evaluated. VX-2 carcinoma not only produced hypercalcemia but also hypophosphatemia and 25-OH-vitamin D deficiency. An increased turnover of 25-OH-vitamin D seems likely. Serum parathyroid hormone and urinary cyclic AMP did not increase. Hypokalemia occurred in association with hypophosphatemia and lowered blood glucose within 1 week after tumor transplantation. At the end of the experiment glucose and insulin were both below the control range. It is concluded that VX-2 carcinoma in rabbits yields much more complex biochemical alterations than reported before on calcium metabolism.