S.C. Brooks

ATM and p21 Cooperate to Suppress Aneuploidy and Subsequent Tumor Development

The DNA damage checkpoint protein kinase mutated in ataxia telangiectasia (ATM) is involved in sensing and transducing DNA damage signals by phosphorylating and activating downstream target proteins that are implicated in the regulation of cell cycle progression and DNA repair. Atm-/- cells are defective in cellular proliferation mediated by the Arf/p53/p21 pathway. In this report, we show that increased expression of p21 (also known as Waf1 or CDKN1a) in Atm-/- cells serves as a cellular defense mechanism to suppress further chromosomal instability (CIN) and tumor development because Atm-/- p21-/- mice are predisposed to carcinomas and sarcomas with intratumoral heterogeneity. It was found that Atm-deficient cells are defective in metaphase-anaphase transition leading to abnormal karyokinesis. Moreover, Atm-/- p21-/- primary embryonic fibroblasts exhibit increased CIN compared with either Atm-/- or p21-/- cells. The increased CIN is manifested at the cellular level by increased chromatid breaks and elevated aneuploid genome in Atm-/- p21-/- cells. Finally, we showed that the role of p21 in a CIN background induced by loss of Atm is to suppress numerical CIN but not structural CIN. Our data suggest that the development of aneuploidy precedes tumor formation and implicates p21 as a major tumor suppressor in a genome instability background.

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.

Characterization of the estrogen receptor transfected MCF10A breast cell line 139B6

SummaryThere has been increasing evidence which suggests that abnormal expression of the estrogen receptor (ER) protein in nonmalignant breast tissue may be important in the carcinogenic process. To examine the effects of ER expression in immortalized nonmalignant mammary epithelial cells, an expression vector containing human ER cDNA was transfected into the ER negative human breast cells, MCF10A. Characterization of a clone stably expressing ER, 139B6, provided evidence for the regulated synthesis of a functional ER capable of binding estradiol-17β (E2) and undergoing processing. Expression of the ER gene did not enable E2 to stimulate endogenous genes [progesterone receptor (PgR), pS2, cathepsin D and TGFα] which normally respond to estrogens in breast cancer cells. The ER in 139B6 cells was, however, capable of inducing expression of an ERE-regulated reporter gene, indicating its ability to interact with transcriptional machinery. Furthermore, cultures in log growth displayed a slight increase in doubling time in the presence of E2. These results indicate that ER expression alone is not sufficient to induce a transformed phenotype. Thus, the 139B6 cell line should provide a new model for determining what additional changes lead to increased growth potential in response to E2 and for exploring how E2 itself may help bring about changes leading to progression of preneoplastic breast epithelial cells.

Induction of tissue plasminogen activator mRNA and activity by structurally altered estrogens

The effect of structure of the estrogen ligand on the accumulation of tPA mRNA and the activity of extracellular fibrinolytic enzyme has been examined in cultures of MCF-7 cells. Estradiol(E2)-stimulated fibrinolytic activity was preceded by an increase in actinomycin D sensitive tPA mRNA synthesis which peaked at 18 h. Ten A- and D-ring structural analogs of E2 affected tPA mRNA accumulation and extracellular fibrinolytic activity. Only in the case of two A-ring isomers (2- and 4-hydroxyestratrien-17 beta-ol) was the decreased effect of the ligands structural change on tPA mRNA accumulation and fibrinolysis not explained by a comparable decline in affinity of the ligand for estrogen receptor. Both of these analogs functioned as antiestrogens. The stimulatory capacity of androstanediols on the tPA gene required that the 3-hydroxyl group be positioned in the beta-configuration. Absence of the 17 beta-hydroxy group was beneficial to the maximum accumulation of tPA mRNA. As has been reported for other estrogen responsive genes (progesterone receptor, cathepsin D and pS2), regulation by estrogens is not related directly to the affinity of the ligand for ER, but this activity may be determined by the location of the electronegative isopotential above the A-ring of estrogenic ligands.

Hepatic sulfation of estrogen metabolites

Abstract 1. 1. Labeled estrone, 17β-estradiol and their sulfates were incubated with rat liver slices and the metabolites were isolated and analyzed according to their conjugated state. 2. 2. The stage of estrous of the rat from which the liver was removed did not influence the hepatic metabolism of the estrogens. The exogenous estrogen 3-sulfate was rapidly hydrolyzed by liver slices, however the added estradiol 17β-sulfate was not hydrolyzed nor was the estrogen nucleus metabolized. The endogenous formation of estrogen 17β-sulfates preceded the synthesis of the estrogen 3,17β-disulfates. 3. 3. Estrone and 17β-estradiol were rapidly intercon verted in incubations with hepatic tissue. The major metabolic pathway observed involved the formation of estrone and its conversion via estrone sulfate and 2-methoxyestrone sulfate to 2-methoxyestrone. The relatively small amount of free 17β-estradiol in these incubations was sulfated at position-17β and ultimately converted to 2-methoxyestradiol 3,17β-disulfate, 2-methoxyestradiol 17β-sulfate, and 6α-hydroxyestradiol 17β-sulfate.

Estrogen receptors and responsiveness of advanced breast cancer to chemotherapy.

We reviewed 89 patients with disseminated breast cancer who had at least one valid estrogen receptor (ER) assay and who underwent one or several trials of chemotherapy. The responses were assessed by two independent extramural reviewers. Of the 89 patients, 81 were evaluable; 28 of 36 (77.8%) ER− positive (ER+) and 28 of 45 (60.7%) ER− negative (ER−) tumors reached at least one remission. Of patients who had received Adriamycin‐containing therapy, 13/20 (65%) ER+ and 12/34 (35.3%) ER− tumors experienced a remission. ER+ breast cancer tends to respond better to Adriamycin‐containing combinations than ER− negative tumors. This study does not support the thesis that lack of estrogen receptor in breast cancer predicts favorably for response to chemotherapy.

Tumor-associated prostaglandins in patients with primary breast cancer: Relationship to clinical parameters

SummaryDetermination of the levels of prostaglandin E2 (PGE2) and PGF2α were carried out using homogenized primary human breast tumors. Measurable levels of both prostaglandins were found in all but one tumor examined. In most samples, the absolute PGF2α level was higher than that of PGE2. Higher PGE2 levels are more often seen in postmenopausal women than in pre- or perimenopausal patients, though among postmenopausal women, PGE2 levels do not correlate with age. Thus, the ratio of PGF:PGE is higher in pre/perimenopausal women than in postmenopausal women. Differences in PGF2α do not appear to be associated with age or menopausal status.Tumors that are estrogen receptor positive (ER +) tend to have higher PGE2 levels than ER negative tumors. PGF2α is not associated with the presence of ER and neither prostaglandin is associated with the presence of progesterone receptor. Higher levels of both PGE2 and PGF2α are associated with less differentiated tumors, while tumor maximum diameter is negatively associated with PGE2 levels.

Skeletal conformations and receptor binding of some 9,11-modified estradiols

The effect of the modification of the 9-11 positions on the skeletal conformation of estradiol (E2) has been analyzed by X-ray crystallography and MM2 molecular mechanics. The 11 beta-hydroxyl and 11-keto analogs of E2 maintained ring conformations which were similar to the natural hormone (E2). Introduction of a double bond at position 9-11 induced a flattening of the entire steroid molecule. An 11 alpha-hydroxyl group brought about significant changes in the alicyclic rings of E2. 9 beta-Estradiol and 11-keto-9 beta-estradiol formed ring conformations which were significantly bent from E2 (below the plane of the A-ring). Examination of the affinity of these C-ring analogs of E2 for the human estrogen receptor has shown extreme variations. A hydroxyl group placed either alpha or beta at the 11-position yielded ligands with vastly different and reduced affinities for the receptor. The low affinity of 11 alpha-hydroxyestradiol (1/300th of E2) may be due to the drastic structural change induced in the alicyclic portion of the molecule, as well as, to the steric or electrostatic effects of the alpha-hydroxyl group upon the receptor protein. An 11 beta-hydroxyl group diminished the receptor binding to 1/60th that of E2 without alicyclic ring distortions, whereas a 9-11 unsaturation reduced the binding to 1/5th although this steroid displayed a flattening of rings B, C, and D. The 11-keto function, which had little effect on the conformation of the estrogen nucleus, reduced the affinity of this ligand to 1/1000th that of E2. The negative bend at the C-ring of 11-keto-9 beta-estradiol and 9 beta-estradiol prevented these ligands from binding receptor. Some of the observed receptor interactions were related to structural alterations in the estrogen ring system induced by modifications on the 9-11 region.

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.