Not only fibroblasts but also melanoma cells express "leucine aminopeptidase" activity.

"Leucine aminopeptidase" (LAP, aminopolypeptidase, EC 3.4.11) activity has been recommended and widely used as a histochemical marker for the identification of contaminating LAP-positive fibroblasts in pigment cell cultures. Using a sensitive biochemical assay with L-leucyl-p-nitroanilide as a substrate we demonstrated that in vitro melanoma cells also exhibit LAP activity. Our comparison of four melanoma cell lines with four fibroblast lines showed that the differences in the enzyme activity were not qualitative but only quantitative. For this reason the specific antibodies, karyological analysis and electron microscopy are recommended as more reliable means in distinguishing fibroblasts from poorly differentiated pigment cells than the LAP-cytochemistry.

Cytotoxic interactions of Zn2+ in vitro: melanoma cells are more susceptible than melanocytes.

Previous studies have shown that sensitivity to high extracellular levels of Zn2+ is a general feature of cells in vitro and that a prerequisite of the toxic action of zinc is entry into cells via channels that are shared with iron or calcium. As the biochemical and toxicological behaviour of zinc chelate complexes could be different from that of free Zn2+, the effect of chelating agents on zinc transport into human melanoma cell lines was tested. EDTAcal and tetracycline reduced the toxic action of zinc ions in vitro, whereas phenytoin and diethyldithiocarbamate potentiated its effects. D-penicillamine, an effective chelator of zinc in vivo, also exerted a protective action in vitro. Comparison of sensitivity to Zn2+ in vitro between human melanoma lines and several lines of pigment cells from skin of various origins demonstrated that melanoma cells are killed by zinc ions at concentrations which are only partially toxic for normal pigment cells. This is consistent with the repeatedly observed high uptake of 65Zn by melanoma cells.

Approaches to prove the melanoma origin in amelanotic human melanoma cell lines.

The human melanoma B-HM8 cell line was derived from highly pigmented malignant skin melanoma. After 5 weeks of cultivation it entirely lost the pigmentation and has remained amelanotic since. Electron microscopy revealed neither premelanosomes nor melanosomes and the cells did not release detectable amount of dopa-oxidase activity into culture medium. Immunocytochemical studies using the polyclonal anti-S-100 antibody and detection of alpha-mannosidase activity in culture medium proved the melanoma origin of B-HM8 cells. Chromosomal changes in the karyotype of these cells were typical for human melanoma with chromosomes No. 1, 5, 7, 9, and 11 involved most frequently.

Acquisition of platinum drug resistance and platinum cross resistance patterns in a panel of human ovarian carcinoma xenografts.

In vivo models of acquired resistance to the platinum-based agents cisplatin (CDDP), carboplatin (CBDCA), iproplatin (CHIP) and tetraplatin have been established using a panel of six parent human ovarian carcinoma lines, two (HX/110 and PXN/87) being derived from previously untreated patients. Resistance has been generated to CDDP (three lines), CBDCA (one line), CHIP (three lines) and tetraplatin (one line) either by treatment in vivo or (for one line to CDDP) through exposure in vitro and subsequent transfer to mice. With the four tumours where resistance was generated using CDDP or CBDCA, a complete cross-resistance to the remaining platinum agents studied was observed. In contrast, in one of three lines with derived resistance to the platinum (IV) agent, CHIP, (PXN/951) a retention in sensitivity was observed with CDDP and CBDCA. Only one of the six parent tumour lines (PXN/100) was markedly sensitive to tetraplatin. Where resistance was generated to tetraplatin (PXN/100T) there was some retention of activity by CDDP. For the CDDP-resistant line established in vitro, there was a close agreement between the cross-resistance profile obtained in vitro vs that obtained in vivo. This tumour panel may be useful in the elucidation of cellular and molecular resistance mechanisms to platinum drugs operative in vivo. Moreover, as they appear to mimic the clinical observations of shared cross-resistance between CDDP, CBDCA and CHIP, they may represent valuable preclinical evaluation models for the discovery of drugs capable of conferring responses in CDDP-refractory ovarian cancer.

Flow cytometry (FCM) of early radiation response in epidermoid carcinoma of uterine cervix.

DNA flow cytometry (FCM) investigation of tumor specimens before and after 30 Gy 137Cs radiation treatment was performed in 33 cases of epidermoid uterine cervix carcinoma. Distinct differences in the type of FCM response to radiation were seen when the results of DNA index (DI) in diploid and aneuploid tumors and proliferation index (PI) values in diploid tumors from pretreatment and 30 Gy irradiated specimens were compared. We observed partial or total reduction of PI in 12 of 17 diploid and near diploid tumors, and total reduction of the aneuploid population in 14 of 16 aneuploid tumors. No significant correlation was found between the type of FCM response and clinical stage of the disease or the histological degree of differentiation.

Tumor progression of murine epidermal cells after treatment in vitro with 12-O-tetradecanoylphorbol-13-acetate or retinoic acid.

Tumor-promoting or antipromoting agents potentially may act directly on initiated squamous epithelial cells or indirectly through effects on normal keratinocytes or immune cells. The purpose of this study was to examine direct effects by comparing in vitro and in vivo treatment of initiated cell populations with 12-O-tetradecanoylphorbol-13-acetate (TPA) or retinoic acid. Keratinocytes were initiated by treatment in vitro with 7,12-dimethylbenz[alpha]anthracene. Replicate cultures of a cloned initiated cell line were exposed to TPA or retinoic acid with acetone as control. After an equivalent number of population doublings, cultured cell sheets were transplanted as skin grafts to athymic nude mice. Replicate grafts from each in vitro treatment group were then treated with TPA or retinoic acid for 8 months. Promotion was quantified by tumor incidence (graft sites with tumor per total sites) and by tumor growth rate. The findings were as follows: (a) TPA increased tumor incidence whether it was applied in vitro or in vivo; (b) TPA in vitro favored more progressive tumors than TPA in vivo; (c) stages of malignant progression from cloned keratinocytes treated in vitro were histologically identical to those following treatment of skin in vivo, including papilloma, dysplastic invasive papilloma, squamous cell carcinoma, and metastasis to lymph node and lung; (d) retinoic acid treatment in vivo reduced tumor incidence and tumor growth rate in initiated cells previously exposed to TPA but not in cells previously exposed to retinoic acid. The results indicated the following: (a) direct effects of TPA on initiated keratinocyte populations were a significant component of tumor promotion; (b) factors in vivo modified the TPA response toward less progressive growth; and (c) the effect of retinoic acid was modulated by prior treatment history.

Flow cytometry (FCM) analysis of endometrial hyperplasia and carcinoma.

In a series of 52 biopsy specimens (31 endometrial carcinomas, 10 atypical endometrial hyperplasias, and 11 cases of normal endometrium), DNA ploidy and S-phase cell fraction were estimated in paraffin-embedded material. DNA aneuploidy was detected in 2 of the 10 atypical endometrial hyperplasias and 7 of the 31 endometrial carcinomas. The majority of aneuploidy was found to be connected with the loss of tumor differentiation. No ploidy disturbances were found in normal endometrium. The S-phase cell fraction value of normal endometrium was significantly lower when compared with that of endometrial carcinoma. The broad variation in S-phase cell fraction values of the endometrial carcinomas and atypical endometrial hyperplasias was in contrast with the low variability of S-phase cell values of normal endometrium. Very low incidence of aneuploidy in the group of well differentiated endometrial carcinomas (Grade I) enables the suggestion that the presence of aneuploidy predicts a more aggressive disease and that the detection of an aneuploid stemline in atypical endometrial hyperplasia may already indicate the neoplastic transformation.

The establishment, characterization and calibration of human ovarian carcinoma xenografts for the evaluation of novel platinum anticancer drugs.

Twenty-three serially transplantable ovarian carcinoma xenografts have been established in female nude mice from forty-two donor samples provided by both previously treated and untreated patients. The lines are all of human karyotype and mirror the histopathology of the original explant. Sixteen examples have been calibrated against four reference platinum drugs: cisplatin, carboplatin, iproplatin, tetraplatin. Three distinct patterns of response were observed: (i) tumours which were comparably sensitive to all four drugs (2 lines); (ii) tumours which were comparably refractory to treatment with all four drugs (5 lines); (iii) tumours which exhibited "individual" patterns of response to the group of calibrating drugs (9 lines). The "response rate" overall of the tumour panel to each of the clinically used drugs was 44% (7/16), though the pattern of response was not identical for each drug. Only 2/16 tumours (13%) were sensitive to tetraplatin, these also being the same tumours which were particularly sensitive to the other three drugs. It was also possible to confirm in 8/9 lines that the therapeutic response of the xenograft reflected that of the corresponding patient's tumour to platinum-based chemotherapy. This tumour panel has now been adopted for evaluating novel platinum-based drugs designed specifically for the treatment of ovarian cancer.

Variation of vascular density within and between tumors of the uterine cervix and its predictive value for radiotherapy.

Vascular density in different regions of a number of uterine cervix carcinomas was determined by morphometric analysis of stained histologic sections. Variance analysis indicated a larger inter- than intra-tumoral inhomogeneity of the vasculature, suggesting a certain individual vascularization pattern in these tumors. As indicated by a retrospective study of archival biopsies, this pattern has a predictive value in regard to the efficacy of radiotherapy.

Biological properties of ten human ovarian carcinoma cell lines: calibration in vitro against four platinum complexes.

Ten human ovarian carcinoma cell lines have been studied as a potential in vitro screen for the development of novel anticancer platinum complexes. Lines have been established and developed both from solid and ascitic tumours, from pretreated and untreated patients, and are available at a range of in vitro passage numbers. The biological properties of the lines were consistent with them being human, epithelial and of ovarian carcinoma origin. Using a tritiated thymidine or leucine uptake method, and a 96 hour continuous drug exposure, the lines have been calibrated against four platinum-containing chemotherapeutic agents: cisplatin, iproplatin, carboplatin and tetraplatin. Striking differences in cytotoxicity were observed across the lines for each agent. Some lines were consistently resistant, others generally sensitive, whereas some showed clear evidence of differential sensitivity to a particular agent. Statistical analysis (Spearman rank correlation) involving the six possible pairings of drugs showed that cisplatin, iproplatin and carboplatin elicit a very similar pattern of response in these lines whereas tetraplatin elicits a completely different response pattern. Similar cytotoxicity values were obtained using a soft agar cloning assay. Results using a tetrazolium dye reduction assay, however, gave somewhat higher and more variable values, particularly with tetraplatin. The thymidine uptake assay will be adopted in further studies on a selected panel of six lines. This panel encompasses the spectra of sensitivities identified for each of the four agents against the original ten lines and may provide a useful screening facility for the development of novel platinum drugs, in that it detects both cell line-determined and structure-determined differences in cytotoxicity.

Flow cytometric analysis of primary cervical and vulvar carcinomas and their metastases.

In a series of 25 patients with cervical carcinoma and 35 patients with vulvar carcinoma, clinically classified (FIGO) as Stage I and Ib, respectively, DNA ploidy and S-phase cell fraction were estimated in paraffin-embedded samples of the primary tumors and their metastases by means of flow cytometry (FCM). The two groups of patients were selected cases in whom lymph nodes removed at radical operation were histopathologically verified as metastatic ones. Prevailing part of primary tumors of both anatomic sites had diploid DNA content and low S-phase fraction. Irrespective of the DNA content and low proliferative activity the early stage carcinomas behaved aggressively as indicated by the presence of metastases, the DNA content of which corresponded in most of the cases with that of primary tumor. It appears that prediction of the tumor growth based only on cytogenetic and cytokinetic parameters may be connected with difficulties.

Vascular density in carcinoma of the uterine cervix and its predictive value for radiotherapy.

The vascular density (VD) in stage-III tumors of the uterine cervix was determined by morphometric analysis of histologic, Masson-trichrome stained sections prepared from biopsies. In a retrospective study, VD was found to be related to results of radiotherapy, larger VD being associated with prolonged survival, in agreement with similar observations made earlier with stage-IB and -IIA tumors of the cervix. In a complementary study the variation of VD within tumors was investigated in relation to the variation between tumors using surgically removed cervical carcinomas in stage IB. Statistically significant F-ratios were calculated, suggesting a more or less individual pattern of vascularization of the tumors despite a considerable intra-tumoral heterogeneity of the vasculature. It is concluded that VD may reflect oxygenation of neoplastic tissue, and may have a predictive value in regard to the response of tumors to irradiation.

Teratological and cytogenetical evaluation of two antihistamines (pipethiadene and pizotifen maleate) in mice.

The teratogenic and cytogenetic effects of two drugs with antihistamine properties, Pipethiadene and Pizotifen maleate, were investigated. Three groups of pregnant mice were treated daily with oral doses (0.24, 0.6 and 1.2 mg/kg) of these drugs from day 4 to day 16 of gestation. The following parameters were investigated: reproductive health of the dams, external, skeletal and visceral malformations of fetuses and frequencies of micronuclei and chromosome aberrations in bone marrow cells of dams. Oral administration of Pipethiadene or Pizotifen maleate produced no teratogenic effects. No elevation was observed in the frequencies of micronuclei and chromosome aberrations. However, the significant reduction of fetal weight after all doses of Pipethiadene or Pizotifen maleate was found to correlate well with the decreased values of the mitotic indices of bone marrow cells of mice, suggesting a potential embryotoxic effect of the tested substances.

Prognostic significance of vascular density and a malignancy grading in radiation treated uterine cervix carcinoma.

In a retrospective study of 95 patients with squamous cell carcinoma of the uterine cervix (Stage IB, IIA, III) treated by radiation only, pretreatment biopsy material was used for assessment of the prognostic value of histopathological multifactorial malignancy grading and a morphometric estimation of vascular density in stroma. By comparison of the two systems, vascular density has been proved superior to malignancy grading with respect to prognostic value.

Immunocytochemical staining of different cell types in vaginal smears by monoclonal anti-DNA methylase antibodies.

Enzymatic methylation of mammalian DNA is closely related to the replication process; also, the synthesis of DNA methylase, an enzyme that is responsible for this process, is cell cycle related. A monoclonal antibody against DNA methylase recognizes proliferatively active cells in the heterogeneous population. We used this antibody to identify the proliferative state of different cell types in normal vaginal smears and in smears of patients with precancerous lesions and cervical cancer. In all preparations, the normal epithelial cells remained unstained. The majority of cancer cells gave a positive immunocytochemical signal indicating the presence of DNA methylase antigen and the proliferative state. Staining was also observed in dyskaryotic cells, particularly in nuclei of parabasal-type cells.

Cytogenetic changes of human peripheral blood lymphocytes in vitro after exposure to cis-DDP (cis-diamminedichloroplatinum II) and oxo-Pt (cis-diamminedichloro-trans-dihydroxyplatinum IV).

Human peripheral blood lymphocytes were tested for induction of micronuclei, chromosome aberrations and sister chromatid exchanges (SCEs) frequency after exposure to two platinum complexes (cis-DDP Platidiam and oxo-Pt) and a comparison was made with the inhibitory effect of these drugs on mitotic activity. When the cis-DDP cell samples were compared with the untreated controls, there was a distinct increase in the frequency of micronuclei and chromosome aberrations, and the statistically significant increase in SCE frequency was accompanied by a significant decrease in mitotic activity. In oxo-Pt cell samples, using an identical concentration of the drug, only a slight increase in micronuclei and chromosome aberration frequency was observed. However, the increase in the SCE frequency was not significant and neither was the decrease of mitotic activity when compared with the controls.