Dwight E. Saunders

Paclitaxel-induced apoptosis in MCF-7 breast-cancer cells

A study of MCF‐7 human breast cancer cells was undertaken to ascertain the degree of apoptosis induction by paclitaxel and if the induction of apoptosis could be enhanced by caffeine. Paclitaxel (0–20 ng/ml) caused concentration‐dependent increases in morphologically identifiable apoptotic cells (up to 43% of cell population) and cells with DNA strand breaks (up to 38%), a commonly cited marker of apoptosis. Maximal DNA strand breakage occurred after 16 hr of exposure to paclitaxel and maximal apoptotic‐appearing cells occurred after 24 hr. The remaining non‐apoptotic paclitaxel‐exposed cells were growth arrested in G2. A 4‐hr exposure to caffeine concentration‐dependently (0–20 mM) increased apoptosis to 88% of the cell population. Our results show induction of apoptosis in breast cancer cells by paclitaxel, and enhancement of this process by caffeine. Int. J. Cancer, 70:214–220, 1997.

Inhibition of c-myc in breast and ovarian carcinoma cells by 1,25-dihydroxyvitamin D3, retinoic acid and dexamethasone.

The role and regulation of the c-myc protooncogene in breast and ovarian neoplasms is receiving increased attention. The downregulation of the c-myc protooncogene by 1,25-dihydroxyvitamin D3 (calcitriol), retinoic acid (RA) and dexamethasone (Dex) is closely associated with growth inhibition in leukemic cells. Calcitriol, RA and Dex have anti-proliferative activity in breast and gynecologic carcinoma cells; however, the regulation of c-myc by these agents in breast and ovarian cancers is mostly unknown. We have addressed the regulation of c-myc in these cancers using an adaptation of a novel method which employs an immunohistochemical procedure to detect c-myc protein followed by quantification of c-myc staining with computerized image analysis. This system represents an alternative to protein product assay by Western blotting and is straightforward, rapid (1 day), can be carried out on a small scale and provides a sample size that readily facilitates statistical analysis of assay data. In MCF-7 human breast cancer cells, c-myc was suppressed 29% by 0.5 nM Dex, 45% by 0.01 nM RA and 54% by 100 nM calcitriol after 24 h of drug treatment. At the same hormone concentrations, growth was inhibited 18% by Dex, 18% by RA and 39% by calcitriol after 3 days of treatment (p < 0.05 for all hormones). Similar patterns of growth and c-myc inhibition were seen in T47D human breast cancer cells and NIH:OVCAR3 human ovarian cancer cells, with the exception of Dex in T47D cells, which caused no inhibition of c-myc or growth.(ABSTRACT TRUNCATED AT 250 WORDS)

The prognostic value of estrogen receptor determinations in patients with primary breast cancer: an update.

Patients with estrogen receptor (E2R)‐positive breast cancers experience a longer disease‐free interval and longer survival following primary surgery than do patients with E2R‐negative tumors. The presence of E2R is correlated with patient age at diagnosis and tumor grade but not with the presence of metastatic foci in axillary lymph nodes. The difference in the rates of recurrence between E2R‐positive and negative tumors is greater in pre‐ and perimenopausal patients than in postmenopausal patients.

Relation of tumor content of estrogen and progesteron receptors with response of patient to endocrine therapy

The levels of estrogen (E2R) and progesterone (PgR) receptors in 165 breast tumor biopsies have been determined. Seventy‐seven percent of the specimens contained significant levels of E2R; 38.2% Were positive for both receptors. Only four tumors displayed the unusual E2R‐/PgR+ pattern. Tumors from pre‐and postmenopausal patients had similar receptor characteristics. Two‐thirds of the six patients with E2R+/PgR+ tumors responded to hormonal therapy, whereas only 28.7% of the E2R+/PgR‐tumors regressed after similar treatment. These data from a limited number of patients indicate that the presence of E2R and PgR in breast tumor biopsies is of higher prognostic value for response of the patient than is the presence of E2R alone.

Receptors for 1,25-dihydroxyvitamin D3 in gynecologic neoplasms

To determine if gynecologic malignancies are candidates for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) therapy we measured vitamin D receptor (VDR) levels in 11 tumor specimens using a radiolabeled ligand-binding assay. VDR was demonstrated in 3 of 6 ovarian tumors and 1 of 1 uterine sarcomas, but not in endometrial tumors (2), cervical tumors (1), or Krukenberg tumors (1). Scatchard plots revealed that [3H]1,25(OH)2D3 was bound to a single class of high-affinity (Kd = 0.3 to 0.6 nM), saturable sites characteristic of authentic 1,25(OH)2D3 receptors. Specificity of binding activity for 1,25(OH)2D3, the active vitamin D3 metabolite, was demonstrated by failure of 25-hydroxy- and 24,25-dihydroxyvitamin D3 to compete effectively against 1,25(OH)2D3 binding in total cellular tumor extracts. The ovarian carcinoma cell line NIH:OVCAR3 was shown to possess VDR (binding capacity = 137 fmol/mg protein, Kd = 0.48 nM). A 3-day incubation of NIH:OVCAR3 cells with 100 nM 1,25(OH)2D3 resulted in 49% inhibition of cell growth. The growth inhibition of an ovarian carcinoma line and the observation that 36% of gynecologic tumors assayed were shown to be VDR-positive suggest that further study is warranted to delineate the mechanism and possible therapeutic aspects of 1,25(OH)2D3 action in gynecologic tumors.

Inhibition of breast and ovarian carcinoma cell growth by 1,25-dihydroxyvitamin D3 combined with retinoic acid or dexamethasone

This study examined the growth inhibitory effects of combining 1,25-dihydroxyvitamin D3 (calcitriol) with retinoic acid or dexamethasone against cultured breast and ovarian carcinoma cells. Retinoic acid (12.5-50 nM) increased the effectiveness of calcitriol (12.5-50 nM) against MCF-7 and NIH:OVCAR3 cells, with synergistic interactions at two of the three ratios tested. Dexamethasone augmented calcitriol effects, with synergism at 0.05 and 0.1 nM dexamethasone in MCF-7 cells and 5 nM in Caov-4 ovarian cells. This study showed favorable interactions for calcitriol-retinoic acid and calcitriol-dexamethasone combinations in breast and ovarian cancer cell lines.

Exposure of embryonic cells to alcohol: Contrasting effects during preimplantation and postimplantation development

Alcohol is a known teratogen that causes a broad variety of developmental anomalies, including fetal growth retardation, craniofacial anomalies, and neurological disorders. The etiology of this multiple defect syndrome, known as fetal alcohol syndrome, has been studied in animal models that reproduce many of the attributes of the human disease. These studies show that ethanol is most teratogenic during organogenesis and development of the nervous system. The molecular basis of fetal alcohol effects has been further investigated using embryo and cell culture systems. Recent studies show that signal transduction pathways controlling cell proliferation are perturbed during ethanol exposure. Ethanol can induce the release of intracellular calcium stores, which stimulates the cell cycle, and it also up-regulates the expression of myc proteins associated with cell proliferation. Increased proliferation is advantageous during the preimplantation period, but ethanol interference with terminal differentiation events within developing tissues during organogenesis may underlie alcohol teratogenicity.

Alcohol inhibits neurite extension and increases N-Myc and c-Myc proteins

Alcohol teratogenesis may be due, in part, to inhibition of neuronal differentiation by alcohol. Because decreases in the N-myc and c-myc proteins are believed to be linked causally to neuronal differentiation, we hypothesized that alcohol would increase N-myc and c-myc proteins in undifferentiated neuronal cells and would oppose the decreases in these two proteins that normally precede differentiation. In undifferentiated LA-N-5 cultured human neuroblastoma cells, alcohol increased N-myc protein levels (178% vs. control cells) and c-myc levels (222% of control). Retinoic acid decreased N-myc and c-myc and induced neurite outgrowth (a differentiation marker). Alcohol prevented retinoic acid-elicited decreases in both myc isoforms and prevented neurite outgrowth. A significant 100% increase in c-myc and an upward trend (48%) in N-myc were observed in CA1 pyramidal neurons of the dorsal hippocampus in mouse fetuses exposed prenatally to alcohol. These data suggest that increases in N-myc and c-myc protein levels are associated with inhibition of neurite extension by alcohol.

Reversal of alcohol's effects on neurite extension and on neuronal GAP43/B50, N-myc, and c-myc protein levels by retinoic acid

Alcohol teratogenesis may be due in part to inhibition of neuronal differentiation by ethanol. We showed previously that alcohol decreased neuronal differentiation (neurite extension) and increased N-myc and c-myc neuronal protein levels. Since Growth-Associated Protein 43 (GAP43/B50) levels must increase for neurons to differentiate, alcohol may decrease GAP43/B50. Alcohol dose-dependently (0-0.5%) decreased GAP43/B50 protein levels by up to 92% in immature LA-N-5 cells. Five nM retinoic acid alone induced differentiation and increased GAP43/B50 levels to 230% of control. These retinoic acid-induced increases in GAP43/B50 and neurite outgrowth, and decreases in N-myc and c-myc, were reversed dose-dependently by alcohol (0-0.5%). Conversely, the adverse effects of 0.25% alcohol on neurite extension, GAP43/B50, N-myc, and c-myc were prevented by 15 and 45 nM retinoic acid. These results suggest that inhibition of neuronal differentiation by alcohol and prevention of such effects by retinoic acid are related to changes in GAP43/B50, N-myc and c-myc.

Clinical application of estrogen receptor in breast cancer.

Patients with ER positive primary tumors usually have initial metastases at the more favorable sites. Twelve out of 14 patients with ER positive had first site of metastases in either bone or soft tissue. In contrast, 13 of 17 patients with negative ER developed first metastases in viscera. ER positive patients respond better to endocrine therapy and survived twice as long as negative ER patients from the onset of recurrent cancer until death. ER content is not a sufficient criterion for the prediction of the response to endocrine manipulation but serves as useful supplementary information to clinical judgement in the selection of systemic therapy. Prior employment of radiation therapy or the administration of hormones or antihormones, as well as inadequate tumor cells in the specimen and poor procurement of the tumor, can produce spuriously low ER. A protocol to study simultaneous endocrine and chemotherapy in comparison to the sequential approach of endocrine treatment followed by chemotherapy in ER positive patients is desirable.