Effect of two superovulation treatments on subsequent fertility in the confined dairy cow.

During a 13-month period, 64 lactating dairy cows of 2 genetic lines, Holstein and crossbred, housed indoors year-round were subjected to 2 superovulations and embryo collections within 112 days post partum. Half of the follicle stimulating hormone (FSH) treatments were given in a descending dosage regimen (Treatment A; 6.5 mg, 5.5 mg, 4.5 mg, and 3.5 mg, twice a day, total 40 mg) over 4 days; the remaining half of the treatments were administered in a constant dosage regimen (Treatment B) of 5 mg twice a day over 4 days. There were no significant differences due to treatment in the number of cows stimulated (more than 2 corpora lutea) nor in the number of ova/embryos collected. However, embryos were obtained from more cows (P<0.05) when treated with the descending dosage regimen. More cows (P<0.05) were stimulated by the superovulatory treatment during the first period than during the second period regardless of the regiment used, treatment A or B. More embryos (P<0.05) were obtained from the Holstein line than from the crossbred line. Fifty-two cows were inseminated at least once after the second embryo collection. Overall, 41 cows (79%) became pregnant after the second collection, requiring up to 4 services. These results suggest that the reproduction of dairy cattle housed indoors year-round is not adversely affected by 2 superovulation treatments and embryo collections within 112 days post partum. The question as to whether the administration of FSH is more efficacious in a descending dosage regimen or a constant dosage regimen was not resolved.

Differential retention of rhodamine 123 by breast carcinoma and normal human mammary tissue.

We have qualitatively evaluated the retention of the fluorescent dye rhodamine 123 by malignant or non-malignant breast epithelial cells in passively-infused fresh surgical specimens. Our findings demonstrate a microscopically-visible increase in the ability of primary and metastatic tumor cells to retain the dye, as compared to non-malignant epithelium. Some variability in fluorescence intensity was seen within and between tumor specimens. The optimal length of incubation in the presence of the dye was critical in achieving differential fluorescence intensity between normal and malignant cells. This method of examining rhodamine 123 uptake and retention in tissue explants provides a reliable means for direct, comparative visualization in situ of any tissue and its associated disorders. The results of this study also demonstrate the validity of extending the use of lipophilic, cationic compounds such as rhodamine 123 as antitumor agents, from model systems to the treatment of malignant disease.

Accessibility to intracellular antigens within nutritionally deprived human mammary epithelial cells.

We have previously demonstrated immunolocalization of antikeratin antibodies in apparently random subpopulations of malignant cells in fresh surgical specimens of breast carcinoma (S. H. Dairkee and A. J. Hackett, 1988, J. Natl. Cancer Inst. 80, 1216-1220). The goal of the present study was to determine whether deficiencies in essential nutrients contribute toward cellular alterations in membrane integrity, consequently allowing antikeratin to bind to the cytoskeleton within live, unfixed cells. We have demonstrated here that in an in vitro model in which human mammary epithelial cells are subjected to an oxygen-glucose gradient, immunolocalization of antikeratin within the cells is observed in a dose-dependent manner in the depleted regions of the gradient, even though the cells appear to be morphologically unaltered. The potential use of antibodies to intracellular antigens for immunotargeting solid tumors and the use of this method in antibody-loading studies toward understanding functional aspects of specific cellular antigens, as well as determining differential response of various cell types under these culture conditions, are discussed.

Preliminary correlations of clinical outcome with in vitro chemosensitivity of second passage human breast cancer cells.

These studies describe the clinical correlations of 63 in vitro chemosensitivity assays on breast cancer cells after short-term monolayer culture. Forty-five of the assays were single agent correlations. Based on cut-off values determined empirically, the test accurately predicted resistance for 36 of 41 patients (88%) who did not respond to the drug. It also predicted sensitivity with a high degree of accuracy: 21 of 22 patients (95%) who responded to the drug tested had a sensitive assay. In five cases, two biopsies were evaluated from the same patient. Whenever assays were performed before and after treatment with a given drug, tumor cells from the second biopsy were more resistant in vitro if the patient failed on therapy. If the patient did not fail, but stopped therapy for other reasons, or if there was no intervening therapy with the tested drug, the two biopsies remained similar in drug sensitivity. These results suggest that in vitro chemosensitivity assays which accurately predict both sensitivity and resistance can be obtained with breast cancer cells after short-term culture and that further prospective trials are warranted.

Internal antigens accessible in breast cancer: implications for tumor targeting.

Proponents of monoclonal antibody (MAb)-mediated cancer therapy often assume that a major limitation in clinical application of MAbs is their lack of absolute specificity for malignant cells. In addition, the presence of surface target antigens is thought to be essential. These requirements may be more stringent than necessary for the clinical usefulness of MAbs. We have demonstrated selective localization of a MAb to keratin polypeptides in malignant breast epithelium under conditions of passive infusion of antibody in fresh surgical specimens of breast carcinoma. Although these proteins are normal intracellular constituents of epithelial cells throughout the body, localization of antikeratin antibodies only within the tumor population is most probably associated with the presence of cells permeable to macromolecules. This permeable tumor cell fraction could be recruited for targeting neighboring impermeable tumor cells with radioisotopes or other antitumor agents conjugated to antibodies directed against intracellular antigens.

Expression of basal and luminal epithelium-specific keratins in normal, benign, and malignant breast tissue.

We characterized the subclass-specific keratins in the epithelium of normal, benign, and malignant breast tissue. Monoclonal antibody 34BE12 stained luminal as well as basal epithelium in normal and benign specimens and all tumor cells in malignant specimens. Antibody 312CS-1 reacted only with basal cells, and antibody LE61 reacted only with luminal cells in the normal and benign specimens. In 34 of 36 breast carcinomas examined, the basal and luminal cell-specific antibodies showed complementary patterns of reactivity, while in the remaining 2 specimens, neither antibody was reactive. The findings reported in this study demonstrate that expression of subclass-specific keratins is mutually exclusive not only in normal and benign mammary specimens but also in breast carcinoma. These findings suggest a role for epithelial subclass-specific antibodies in the histogenetic and prognostic subclassifications of breast carcinoma.

Immortalization in culture: occurrence at a late stage in the progression of breast cancer.

The properties in culture of 3 breast cancer effusion metastases, obtained over approximately 2 years from the same patient, were examined. Despite repeated attempts with cryopreserved cells, only the last specimen reproducibly exhibited immortality in culture; the first 2 specimens grew initially but failed to develop into cell lines. Each specimen was unique in morphology and growth properties, although karyotypic markers indicated a common origin. Aberrations of chromosomes 1 and 11 marked these near-diploid cells, and further structural alterations of chromosome 11 accompanied the transition of biological properties observed in the third specimen.

Use of cells cultured from human mammary carcinomas for studies of malignant progression and chemotherapeutic drug sensitivity.

We describe techniques to readily culture human mammary epithelium from normal and malignant tissue. The cells were characterized as to their epithelial origins and qualities distinguishing tumor from normal cells. Using a highly efficient clonogenic assay, we now are able to routinely assay breast tumors for chemotherapeutic drugs. Initial studies suggest that these assays correlate with patient response.

Testosterone of estradiol treatment of heifers or freemartins to detect estrus in confined dairy cattle.

Dairy heifers and freemartins were injected with either testosterone or estradiol and then used to detect estrus in mature dairy cows maintained indoors year-round on slatted-concrete floors. During a 15-mo period, 1,026 occurrences of estrus were confirmed from 339 cows. Observations by the herdsmen detected 92% of all occurrences of estrus compared to the heifers (39%) and freemartins (23%). There was great variation among animals in their ability to detect estrus. Conception rates were not affected by the method of detection of estrus.

Use of enzyme-linked immunosorbent assay for measurement of bovine serum and milk progesterone without extraction.

An enzyme immunoassay for progesterone, using horseradish peroxidase as the label, was adapted for direct measurement of progesterone in serum or milk. Values obtained by direct measurement are highly correlated with values measured in extracts and are usable for convenient, rapid, accurate monitoring of the reproductive status of dairy cows. The assay is sensitive (ca. 1 pg), rapidly performed (3.5 h), and allows 92% accuracy in assessment of pregnancy status by direct measurement of progesterone in paired milk samples collected at breeding and 21 d later.

Concurrent expression of basal and luminal epithelial markers in cultures of normal human breast analyzed using monoclonal antibodies.

The aim of this study was to investigate the retention in culture of the antigens characteristic of the two mammary epithelial subclasses, basal and luminal epithelium. Primary and secondary cultures of normal human mammary-gland cells were used for immunolocalization experiments with monoclonal antibodies to luminal and basal epithelium. In contrast to the in vivo situation, in which reactivity was only seen in basal cells that were negative for the luminal antigen, we found the homogeneous expression of the basal marker by all of the cultured cells at second passage, and the simultaneous expression of the luminal marker by some of these cells. Characterization of the basal antigen expressed in culture using sodium-dodecyl-sulfate/polyacrylamide gel electrophoresis and immunoblotting techniques showed it to be a 51-kilodalton keratin peptide with an isoelectric pH of 5.4, and confirmed its similarity to the antigen expressed in vivo. Our findings thus demonstrated the coordinate expression of the basal and luminal antigens in cells cultured on solid substrates. The availability of monoclonal antibodies to epithelial-subclass-specific markers of the human mammary gland now makes it feasible to search for culture conditions that would allow the maintenance and manipulation of cell differentiation in vitro.

Monoclonal antibody that defines human myoepithelium.

We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.

Early expression of vimentin in human mammary cultures.

The intermediate filaments of most epithelial cells in vivo consist solely of cytokeratins. Using monoclonal antibodies to vimentin or keratin, we have examined the expression of vimentin in homologous specimens of frozen tissue sections and primary cultures of normal human mammary epithelium. In frozen sections, only epithelial cells reacted with the antikeratin antibody, whereas antivimentin reactivity was associated with stromal cells. All epithelial cultures were positive for cytokeratin and in addition coexpressed vimentin as strongly as cultured fibroblasts and as early as the 4th d after initiation of the culture. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of cytoskeletal preparations of secondary cultures of normal mammary epithelium have also demonstrated the appearance of a moiety identical to the vimentin found in cultured fibroblasts. Our observations are consistent with the hypothesis that vimentin expression is induced, possibly as a result of changes in cell shape or growth rate, when cells are freed from three-dimensional restrictions imposed by the tissue of origin.

Growth of diploid cells from breast cancers.

Cell cultures were derived from normal and cancerous breast tissues and from metastases by methods that selected for relatively adherent epithelial aggregates. Karyotypic analyses of first or second passage cultures yielded predominantly normal diploid cells. Nonclonal aberrations were more common in tumor-derived than in normal cultures. Three of the cultures that originated from metastases were characterized by abnormal clones. These results support observations based on DNA content, which indicate that a considerable fraction of breast cancers are composed predominantly of diploid cells. They differ greatly from chromosomal findings in long-term cultures of tumor effusions and thus emphasize the karyotypic diversity that can be found in tumors from a single tissue of origin--the breast.

Invasiveness and ploidy of human mammary carcinomas in short-term culture.

Invasiveness and ploidy were examined in cultures of human epithelial cells derived from nonmalignant breast tissue, primary breast carcinomas, and breast cancer effusion metastases. Successful short-term culture was achieved from approximately 70% of the primary breast cancers. These primary cancers were essentially diploid by flow cytometry and karyotype in contrast to the effusion metastases, which were mostly aneuploid. The diploid tumor cells retained their malignant phenotype in culture as demonstrated by invasion into a denuded human amnion basement membrane. In contrast, epithelial cells cultured from nonmalignant mammary tissue did not invade the amnion. We suggest that the diploid carcinoma cultures may be useful for investigating the essential differences between normal and malignant cells and may complement information derived from studies of tumor cell lines with grossly aberrant karyotypes.

Response to doxorubicin of cultured normal and cancerous human mammary epithelial cells.

Epithelial cells were isolated and cultured from a number of human mammary specimens of both cancerous and noncancerous origin. Doxorubicin (Dx) sensitivity was measured at second passage with the use of a highly efficient clonogenic assay. For 23 different tumor specimens derived from patients without previous chemotherapy, the drug concentrations required to kill 50% of the cells varied approximately 35-fold. In contrast, for 11 tumor specimens from patients who relapsed after regimens containing Dx, the drug concentration for 50% survival varied only fivefold and the dose-response curves for these specimens clustered at the more resistant end of the spectrum. A wide range of sensitivities was also observed among 13 noncancerous mammary specimens; however, tumor tissue and noncancerous tissue from the same donor were similar. When cultures were subjected to drug incubation periods of 1 and 4 hours, dose-response curves were superimposable when plotted as a function of drug concentration multiplied by time.

Reproductive performance of Finnish Landrace and Suffolk sheep maintained indoors year-round.

The reproductive performance (fertility and prolificacy) of Finnish Landrace (Finn) and Suffolk sheep on an accelerated breeding program was evaluated. Both breeds were contained in each of two separate flocks housed indoors year-round on expanded metal floors in windowless buildings. The two flocks were bred alternately at 4-mo intervals in January, May and September. The sheep were exposed to either an abrupt (ALR) or constant (CLR) lighting regimen. Data from nine breedings during a 4-yr period were considered. Fertility was significantly higher for ewes in the ALR lighting regimen. Fertility was lower for ewes bred in September. For ewes that had lambed from breeding 8 mo earlier, fertility was higher for Finn ewes than for Suffolk ewes; however, similar fertility levels were observed in both breeds if the ewes had not lambed following the previous breeding. Higher prolificacy was observed in the Finn ewes than in the Suffolk ewes, but the differences in prolificacy varied with the month of breeding. The probability of a ewe having more than one lamb was significantly higher for Finn ewes than for Suffolk ewes in both January and May breedings, but was similar in September breedings. Incorporating the Finnish Landrace breed into an accelerated breeding program for sheep maintained indoors year-round is a practical way to increase the number of lambs born. Controlling daylength and dividing the flock into smaller flocks are also practical procedures to increase lamb production.