Biological activity of all-trans-retinoic acid with and without tamoxifen and alpha-interferon 2a in breast cancer patients.

In addition to suppressing breast cancer cell growth, retinoids potentiate growth inhibition in human breast cancer when tested in vitro and in vivo with tamoxifen and/or interferon. The purpose of this study was to ascertain the biologic effects of all-trans-retinoic acid (ATRA) administered alone and with tamoxifen +/- interferon and to identify the relationship between ATRA plasma concentrations and optimal biological dose (the lowest dose that produces a biological response). Three consecutive groups of 15 patients with locally advanced operable breast cancer were treated, in accordance with good clinical practice (GCP) requirements, with ATRA at 3 dose levels alone or with tamoxifen +/- alpha-interferon 2a at flat doses. After 3 weeks, the tumors were surgically removed. Biological parameters measured at the beginning (in biopsy tissue) and end (in surgical tissue) of the study were compared. The optimal biological dose for ATRA was 15 mg/m2/day. Treatments influenced tumor grade but not cell cycle kinetics (G0-G1 phase) or proliferation (Ki67 levels). ATRA induced progesterone receptors independent of dose level and co-administered drugs, but did not induce estrogen receptors when administered alone. Retinoic acid receptor (RAR)-alpha was not affected by treatment and RAR-alpha was moderately influenced whereas RAR-beta (concomitantly with transforming growth factor-beta) was induced in 33% of patients by ATRA alone. ATRA pharmacokinetics were dose- and time-dependent. Neither the ATRA + tamoxifen nor the ATRA + tamoxifen + interferon combinations potentiated the ATRA-induced biological changes. Future studies evaluating the role of RAR-beta as a biological marker of retinoid activity are warranted.

Cytofluorometric and cytogenetic evaluation of renal cell carcinoma: correlations with the clinical course.

The tumor specimens of 20 patients who underwent radical nephrectomy for nonfamiliar unifocal renal cell carcinoma were evaluated with conventional histopathology, cytogenetic analysis and evaluation of nuclear ploidy with cytofluorometry. The pathological stages were 1 T1N0M0, 10 T2N0M0, 1 T2N + M0, 6 T3N0M0 and 2 T3N + M0. Eleven tumors presented clonal chromosomal abnormalities, 2 had chromosomal instability and 7 normal karyotype. Rearrangement of chromosome 3 was documented in 2 cases only, in contrast with previous observations in the literature. Ten patients had a diploid or peridiploid DNA tumor content, 1 tetraploid, 4 triploid and 5 multiclonal. No correlation was found between nuclear ploidy and karyotype. Chromosomal abnormalities did not impact on survival, while a statistically significant correlation was found with diploid nuclear DNA content, since with a mean follow-up of 45.7 months, 9 out of 10 patients with diploid and peridiploid tumors are alive (p < 0.02), while 8 out of 10 nondiploid died of disease progression.

Age-related changes in neutral lipid content of Paramecium primaurelia as revealed by nile red.

Cellular neutral lipid content of Paramecium primaurelia was measured during culture and clonal life using nile red (9-diethylamino-5H-benzo[alpha] phenoxazine-5-one), a dye utilized for lipid analysis in both mammalian cells and in ciliated protozoa. Lipid droplets in P. primaurelia are concentrated in the anterior pole of the cell; their number is maximum in early log phase cells and decreases in late log phase cells. The quantitative determination of neutral lipids was obtained measuring the fluorescence from the excitation and emission spectra of 480 nm and 540 nm, respectively. Neutral lipid content decreases linearly during the log phase of growth while the decline is minimum during the stationary phase. In the late log phase, the amount is 30% of that of the early log-phase cells. Though the cell size declines too, cell area and lipid content decreases are not correlated in the middle log phase, because the maximum lipid reduction is obtained when the cell size is relatively constant. The cellular lipid content also changes during the clonal life. Neutral lipids decrease discontinuously (r = -0.75, P < 0.05) as the fission age increases. No relationship was found between lipid content and food vacuole formation during both culture and clonal life.

Cytofluorometric evaluation of nuclear DNA content distribution in renal neoplasms treated by conservative surgery.

The prognostic value of nuclear DNA content in 21 patients who underwent conservative surgery for renal tumors was the object of a retrospective study. Cytofluorometric evaluation of nuclear DNA content distribution was performed on smears obtained from 50-micron thick histologic sections prepared with the Hedley technique, using a Leitz MPVII microspectrophotometer. The DNA indexes were plotted in the form of frequency histograms. DNA measurements were repeated by a cell image analysis system (CAS 200; Becton Dickinson) in 16/21 cases. Nuclear DNA content was diploid in 12 cases, triploid in 4, tetraploid in 2 and multiclonal in 3. No statistically significant correlation was found between ploidy and tumor stage and size, using Kendall's tau test, while there was a significant correlation between tumor ploidy and nuclear grade (p < 0.01). Excluding 2 postoperative deaths, with an average follow-up of 54 months, tumor diffusion was observed in 2 patients (1 with multiclonal and 1 with triploid DNA content) and a local recurrence in a patient with a triploid tumor. Of the remaining 16 no evidence of disease (NED) cases, 10 have a diploid DNA tumor content, 2 diploid, 2 tetraploid and 2 multiclonal. It is concluded that even after conservative surgery for renal neoplasms a diploid DNA content is a favorable prognostic factor. A completely negative prognostic impact of nondiploid tumors is not confirmed, since although all three negative events occurred in this group, there are also 4 NED patients with respectively 70, 46, 43 and 40 months' follow-up.

A study on macronuclear chromatin structure in Colpoda inflata (Protozoa, Ciliophora) by means of image analysis.

Resting encystment is a reversible cytodifferentiation phenomenon regularly taking place during the life cycle of Colpodidae. The encystment involves regulation of the macronuclear DNA content, accomplished via chromatin extrusion and DNA synthesis mechanisms. An analysis of macronuclear chromatin texture was carried out in Colpoda inflata logarithmically growing cells, pre-cystic cells, and increasingly aging resting cysts after Feulgen staining. Morphometric and densitometric parameters as well as Markovian chromatin texture variables were utilised for image analysis. Moreover, univariate and multivariate analyses were applied both to macronuclei and to their chromatin extrusion bodies. The results show that structural variations in chromatin appear under macronuclear functional activity conditions, suggesting a dynamic state of chromatin texture throughout a long-term resting encystment.

Evaluation of cathepsin D as prognostic predictor in breast cancer.

Cathepsin D is an acidic lysosomal protease expressed in all cells. Some studies have shown correlations between high levels of tissue cathepsin D and poor prognosis. This paper deals with 158 cases of breast cancer in which tissue concentrations in cathepsin D, age, estrogen and progesterone receptor content, and pathological characteristics of the tumor were investigated. Tumors were considered to be cathepsin D+ when a concentration > 40 pmol/mg protein (median value in our samples) was determined. The expression of cathepsin D appears to be related to grading (p = 0.04) and lymph node status (p = 0.05). We found no significant associations among cathepsin D levels, patient age, steroid receptors and histological type. Moreover, the levels of cathepsin D have been evaluated in 9 samples of recurring or metastatic neoplasia and 11 cases of benign breast lesions. We conclude that cathepsin D may be a useful prognostic predictor in breast cancer. Further investigations are required to improve and extend the applications of this assay.

Cytophotometric analysis of the nuclear uptake of adriamycin in in vivo normal mouse liver and kidney cells.

Adriamycin levels are usually determined in biologic samples (tissue homogenates) by means of spectrophotofluorimetric procedures. After intravenous administration of a pharmacologic dose of Adriamycin to 21 female Swiss mice, the fluorescence released from the nuclei of single liver and kidney cells was determined by cytofluorimetric measurements on 3,916 in-vivo-treated liver cells and 1,472 in-vivo-treated kidney cells. Similar measurements were made on 287 untreated (control) liver cells and 288 untreated kidney cells as well as on 541 liver cells and 773 kidney cells stained in vitro with acriflavine. The results indicate that (1) it is possible to quantitatively evaluate the nuclear uptake of Adriamycin in in vivo single cells by cytofluorimetry, (2) the pharmacokinetics of Adriamycin can be evaluated on the cellular level and (3) Adriamycin can be considered a fluorochrome suitable for in vivo staining of cell nuclei as can acriflavine for the in vitro staining of cell nuclei.