E. Schauenstein

Über die weitgehend selektive Abtötung subcutan implantierter Ehrlich-Ascites-Tumorzellen in vivo durch 4-Hydroxy-enale, insbesondere 4-Hydroxy-pentenal

The effects of homologue 4-hydroxy-enales, especially 4-hydroxy-pentenale (HPE), on subcutaneously implanted Ehrlich-ascites-tumor-cells (EATC) in vivo, and the persistence of HPE in the subcutaneous tissue have been investigated. After “incubation” of EATC in subcutaneous depots of HPE, all of the tested hydroxy-enales caused death of about 90% of the EATC, as indicated by positive reaction with trypan-blue. Hydroxy-hexenale turned out to be less effective. However, no distinct influence of the length of the hydrocarbon-chain upon the effectiveness of the aldehydes was observed. HPE injected simultaneously with EATC into subcutaneous tissue showed, depending on the given dose, an optimal inhibitory effect at about 3·10−7 m/g body weight. Three weeks after implantation, 50% of the animals remained tumor-free, 48% showed predominantly strong inhibition of the tumor-growth, and 2% showed no significant inhibition. The average inhibition of tumor-growth was 91%. Twelve weeks after the same treatment 50% of the treated animals also remained tumor-free. Measurements of the persistence of HPE in the subcutaneous tissue showed, depending on the injected dose, an optimum of about 2.5–3.5·10−7m/g body weight. The half-life of the free HPE in the subcutaneous tissue came to 2–3 minutes. Es wurden die Wirkung von vier homologen Hydroxy-enalen, insbesondere von Hydroxy-pentenal, auf subcutan implantierte Ehrlich-Ascites-Tumorzellen in vivo sowie die Persistenz von Hydroxy-pentenal in subcutanen Depots untersucht. Dabei ergab sich: Alle untersuchten Hydroxy-enale führten bei “Inkubation” von EATZ in Subcutandepots und anschließender Färbung mit Trypanblau zu etwa 90% trypanpositiven, also sicher devitalisierten Zellen. Ein gesetzmäßiger Gang der Abhängigkeit der Wirkung von der Kettenlänge der Aldehyde konnte nicht nachgewiesen werden. Die Simultanversuche ergaben abhängig von der gegebenen Dosis ein Wirkungsoptimum von HPE bei 3·10−7m/g KG nach 3 Wochen, wobei bei 50% der behandelten Versuchstiere das Tumorwachstum verhindert, bei 48% verzögert und bei 2% ungehemmt war. Die mittlere Hemmung des Tumorwachstums betrug 91%. Der zwölfwöchige Simultanversuch bestätigte die oben genannten Ergebnisse: 10 von 20 Versuchstieren überlebten die Beobachtungszeit tumorfrei. Persistenzmessungen an HPE in subcutanen Depots ergaben ein Maximum der Verweilzeit des Aldehyds im Bereich von 2,5–3,5·10−7m/g KG. Die Halbwertszeit für das freie HPE im subcutanen Gewebe beträgt 2–3 min.

Quantitative cytospectrophotometric studies on protein thiols and reactive protein disulphides in samples of normal human uterine cervix and on samples obtained from patients with dysplasia or carcinoma-in-situ.

Quantitative microspectophotometric studies have been made on sections of human cervix after staining for reactive protein thiol-groups (PSHr), and the sum of protein thiols with so-called reactive protein disulphides (together abbreviated as TRPS). Measurements were made on normal epithelium, apparently normal epithelium adjacent to a pathological lesion, dysplastic epithelium, carcinoma-in-situ, and adjoining stroma. The numbers of cases studied were: normal healthy controls (53); patients with dysplasias (34) and patients with carcinoma-in-situ (29). In the normal control sections the ratio of PSHr in epithelium:stroma was approximately 2.7 and this ratio was strongly decreased in dysplasias (1.6) and carcinoma-in-situ (1.5); the 3 populations of values had sufficient overlap to prevent this measurement being an effective discriminator. No significant variations were observed with TRPS-values except with changes in the stroma adjacent to apparently normal epithelium. However, the ratio of PSHr:TRPS was effectively discriminatory when this double-staining ratio was calculated for epithelial values:stromal values. These results are discussed in relation to the importance of thiol-groups in cell division and cancer, and the biological implications of similar changes observed in neighbouring apparently normal epithelium.

Therapieversuche mit Hydroxypentenal am Ehrlich Ascites-Solidtumor der Maus

Subcutaneous peritumoral injections of 4·10−7 moles of 4-hydroxy-2-enal-1 (HPE), given as a doublet of 2 single doses of 2·10−7 moles at intervals of 15 minutes completely destroy the 3-day-Ehrlich solid tumor of mice macroscopically in 31% of test animals, inhibit the growth of the tumor considerably in 49.6% and produce no essential effect in 19.4%. The therapeutic index is 0.23, representing a significant and good tumor inhibition. The same dosage gives a therapeutic index of 0.26 if applied to the 8-day-tumor. If the dosage is raised to 8·10−7 moles per g b. w. (2 doublets of 4·10−7 moles at intervals of 3 days, single doses 2·10−7 moles at intervals of 15 minutes), a macroscopically complete destruction of the 8-day-tumor is observed in 35% of the animals, a considerable inhibition in 61% and essentially no influence in 4%. The therapeutic index is 0.23; time of observation in all series 3 weeks. Healthy control mice receiving only therapeutic doses showed no mortality, whereas the loss of animals in the therapy series was about 10%. Of the treated animals, 80% showed local skin defects (⊘ 0.5–1 cm) at the injection site which healed up promptly without complications. The extremely short persistence of the HPE in the subcutaneous area leads to the assumption that the effective dose is much lower than the one actually given. Die therapeutische Wirkung von 4-Hydroxy-2-enal-1 (HPE) auf den Ehrlich-Solidtumor der Maus wird untersucht. Bei subkutaner peritumoraler Injektion von 4·10−7 Molen HPE pro g KG als Doppelgabe von je 2·10−7 Molen im Abstand von 15 Minuten wird der 3 Tage angegangene Tumor in 31% der Fälle makroskopisch vernichtet, in 49,6% im Wachstum weitgehend gehemmt und in 19,4% nicht ins Gewicht fallend beeinflußt. Therapie-Index 0,23, was einer deutlichen (guten) Tumorhemmung entspricht. Beim 8-Tage-Tumor bringt die gleiche Dosierung eine etwas ungünstigere Wirkung (Therapie-Index 0,26). Mit 8·10−7 Molen HPE per g KG, in 2 Doppelgaben von je 4·10−7 Molen, am 9. und 12. Tag appliziert, wird in 35% der Fälle eine makroskopisch vollständige Verhinderung, in 61% eine weitgehende Verzögerung und in 4% keine wesentliche Beeinflussung des Tumorwachstums festgestellt. Therapie-Index 0,23. Beobachtungsdauer durchweg 3 Wochen. Die mitgeführten Verträglichkeitskontrollen zeigten keine, die Therapie-Serien ca. 10% Verluste. Bei den Therapietieren traten in 80% an der Injektionsstelle lokale Hautschäden (⊘ 0,5–1 cm) auf, die unter glatter Narbenbildung abheilen. Die außerordentlich kurze Persistenz des HPE im Subkutanbereich läßt annehmen, daß die wirksamen Dosen weit niedriger liegen dürften als die tatsächlich verabreichten.

Histophotometrical investigations on the contents of protein and protein thiols of the epithelium and stroma of the human cervix

SummarySections of 10 μm thickness of normal epithelium of the cervix uteri were stained with DDD-Fast Blue B for reactive protein thiols (PSHr) and with Amidoblack for proteins. Areas of approximately 0.3 mm2, both of the intact epithelium and of the adjacent stroma were scanned at 560 and 620 nm respectively, using a measuring spot of 10 μm ∅ and the average densities per unit area (E/μm2), of PSHr and proteins, were determined. No essential differences were found between the four different cell layers (basal (B), parabasal (P), intermediate (I) and superficial (S)) either with respect to PSHr or to the protein area densities. Thus it seems to be justified to introduce E/μm2-values for the epithelium as a total: 0.33 for PSHr and 0.40 for protein and to compare them with the corresponding values of the stroma: 0.13 and 0.23 respectively. The epithelium contains approximately 2.5 times more PSHr but only 1.7 times more proteins than the adjacent stroma, indicating that the greater PSHr-content of the epithelium is caused not only by the greater content of epithelial proteins but also by the greater PSHr-content of the epithelial proteins. The approximately equal densities per unit area (E/μm2) of the PSHr in the four epithelial cell layers, however, do not give any information about the intracellular localisation of PSHr. The investigation of individual single cells in the sections with a resolution of a 1 μm-∅ measuring spot shows that in the actively proliferating B- and P-cells nuclear PSHr make up 60 and 80% respectively of the total PSHr present, but only 5% in the non-dividing S-cells where considerable percentages of PSHr were found not only in the cytoplasm (46%) but also in the outer cell membrane (28%) and in the intercellular substance (22%). In summary, it can be said that the higher content of PSHr in the epithelium compared with the adjacent stoma points to a greater extent of the SH-controlled processes in the epithelium; these might be processes in connection with cell proliferation in the stratum germinativum, whereas in the superficial cell layer, functions of the outer cell membrane and of the intercellular substance might be relatively more important.

Quantifizierung der Proteinthiole in morphologisch normalen Basalzellen und pathologischen Platten-Epithelzellen der Cervix Uteri

Smears taken from eight probands with carcinoma in situ or invasive carcinoma of the cervix uteri have been stained with DDD and Fast Blue B. The extinctions microspectrometrically measured at 560 nm are directly proportional to the quantity of protein-SH-groups. The extinctions of the total cell (Eges) and of the cell nucleus (EK) are measured in 67 basal cells (BAS), 78 dysplatic cells (DYS), 122 undifferentiated cancer cells (UNIF) and 89 differentiated cancer cells (POLY). From BAS through DYS and UNIF to POLY EK increases by a total of 176%. In all four cell types investigated, linear correlations between EK and Eges have been found to occur with a probability of over 99%. The straight lines ascertained represent a relation between EK and Eges which is obviously very characteristic for each cell type, and it becomes apparent that the measuring points corresponding to each single cell are in each instance so close to the straight line that in most cases a differentiation of the three pathological cell types is possible even without a morphological criterion. The straight lines corresponding to BAS, DYS and UNIF start from a common origin, whereas the straight line corresponding to POLY branches off from the UNIF line only. This is in accordance with the formal genesis of pathological variants observed in the cervical squamous epithelium or in differentiated carcinomas of the squamous epithelium respectively.Smears taken from eight probands with carcinoma in situ or invasive carcinoma of the cervix uteri have been stained with DDD and Fast Blue B. The extinctions microspectrometrically measured at 560 nm are directly proportional to the quantity of protein-SH-groups. The extinctions of the total cell (E ges) and of the cell nucleus (E k) are measured in 67 basal cells (BAS), 78 dysplastic cells (DYS), 122 undifferentiated cancer cells (UNIF) and 89 differentiated cancer cells (POLY). From BAS through DYS and UNIF to POLY E k increases by a total of 176%. In all four cell types investigated, linear correlations between E k and E ges have been found to occur with a probability of over 99%. The straight lines ascertained represent a relation between E k and E ges which is obviously very characteristic for each cell type, and it becomes apparent that the measuring points corresponding to each single cell are in each instance so close to the straight line that in most cases a differentiation of the three pathological cell types is possible even without a morphological criterion. The straight lines corresponding to BAS, DYS and UNIF start from a common origin, whereas the straight line corresponding to POLY branches off from the UNIF line only. This is in accordance with the formal genesis of pathological variants observed in the cervical squamous epithelium or in differentiated carcinomas of the squamous epithelium respectively. Zellabstriche von 8 Patientinnen mit Carcinoma in situ bzw. invasivem Carcinoma der Cervix uteri wurden mit DDD-Echtblau B gefärbt. Die bei 560 nm mikrospektrometrisch gemessenen Extinktionen sind der Menge an Protein-SH-Gruppen direkt proportional. Die Extinktionen der gesamten Zellen (Eges) und der Zellkerne (E k) wurden an 67 Basai (BAS), 78 dysplastischen (DYS), 122 uniform-atypischen (UNIF) und 89 polymorphatypischen (POLY) Zellen gemessen. E k nimmt von BAS über DYS und UNIF zu POLY um insgesamt 176% zu. Bei allen 4 untersuchten Zelltypen ergaben sich mit über 99% Wahrschein lichkeit lineare Korrelationen zwischen E k und E ges. Die berechneten Geraden repräsentieren ein für jede Zelltype offenbar sehr charakteristisches Verhältnis von E k zu E ges und es zeigt sich, daß die einzelnen Zellen entsprechenden Meßpunkte jeweils so dicht an den Geraden liegen, daß eine Unterscheidung der 3 pathologischen Zelltypen in den meisten Fällen auch ohne morphologisches Kriterium möglich wird. Die den BAS, DYS und UNIF entsprechenden Geraden gehen von einem gemeinsamen Ursprung aus, während die den POLY zugehörige Gerade erst von der UNIF-Geraden abzweigt. Dies steht in Übereinstimmung zur formalen Genese pathologischer Varianten des zervikalen Plattenepithels bzw. differenzierter Plattenepithelkarzinome.

Histophotometric quantification of protein and reactive proteinthiols in invasive carcinomas of the skin.

SummaryFrozen sections cut from 14 samples of invasive carcinomas of the skin were stained with Amido black for protein determination and with dihydroxydinaphthyldisulphide fast blue to quantify reactive protein thiols (PSHr) and were then analysed microphotometrically. It was found that all of the samples exhibited significant reductions in protein levels (49%–74%) and PSHr levels (32%–53%) as compared to normal epidermis. Thus, the content of proteins of PSHr groups was 1.7 times greater in the malignant tissue examined than in normal epidermis.These results are in accordance with those previously obtained in basal-cell epitheliomas.