Estrogen receptor-positive mammary tumorigenesis in TGFalpha transgenic mice progresses with progesterone receptor loss.

We characterized the novel NRL-transforming growth factor alpha (NRL-TGFalpha) transgenic mouse model in which growth factor - steroid receptor interactions were explored. The NRL promoter directs transgene expression to mammary ductal and alveolar cells and is nonresponsive to estrogen manipulations in vitro and in vivo. NRL-TGFalpha mice acquire proliferative hyperplasias as well as cystic and solid tumors. Quantitative transcript analysis revealed a progressive decrease in estrogen receptor alpha (ER) and progesterone receptor (PR) mRNA levels with tumorigenesis. However, ER protein was evident in all lesion types and in surrounding stromal cells using immunohistochemistry. PR protein was identified in normal epithelial cells and in very few cells of small epithelial hyperplasias, but never in stromal or tumor cells. Prophylactic ovariectomy significantly delayed tumor development and decreased incidence. Finally, while heterozygous (+/-) p53 mice did not acquire mammary lesions, p53+/- mice carrying the NRL-TGFalpha transgene developed ER negative/PR negative undifferentiated carcinomas. These data demonstrate that unregulated TGFalpha expression in the mammary gland leads to oncogenesis that is dependent on ovarian steroids early in tumorigenesis. Resulting tumors resemble a clinical phenotype of ER+/PR-, and when combined with a heterozygous p53 genotype, ER-/PR-.

Dysregulation of mammary Stats 1,3 and 5 and PRL receptors by overexpression of TGFalpha.

Mammary TGFalpha overexpression results in delayed involution and eventually mammary cancer in transgenic mice. We hypothesized that STATs and PRL receptors (PRLR), critical regulators of mammary function, are altered in these animals and may contribute to this phenotype. We examined these factors late in the first pregnancy (d.18) and during normal involution (d.4 post-lactation) in WAP-TGFalpha transgenic mice and non-transgenic controls. Long form PRLR mRNA in WAP-TGFalpha glands at both pregnant d.18 and d.4 post-lactation was significantly reduced compared to controls, and PRLR-S3 failed to rise during involution. Total and pTyr STAT 1,3,5a and 5b also were altered. STAT 3 was higher at both times in WAP-TGFalpha glands. STAT 5a and 5b were lower at late pregnancy, but higher post-lactation; however, pTyr(694) STAT 5 was abnormally low at both times. Thus overexpression of TGFalpha has direct or indirect effects on both STATs and PRL responsiveness in vivo, which may reflect mechanisms of TGFalpha-induced mammary epithelial abnormalities.

Casein kinase I alpha and alpha L: alternative splicing-generated kinases exhibit different catalytic properties.

Casein kinase I (CKI) is a family of serine/threonine protein kinases found in all eukaryotes examined to date. Here, the rat CKI isoforms alpha and alpha L were cloned and expressed in both eukaryotic and prokaryotic systems. Characterization of the genomic DNA flanking the exon unique to CKI alpha L demonstrated that CKI alpha and CKI alpha L arise by the alternative splicing of a common pre-mRNA molecule. To the best of our knowledge, the alpha L isoform is the only known active serine/threonine kinase to contain an insert within its catalytic domain. Tissue distribution of each splicing isoform was examined by RT-PCR, immunoprecipitation, and Western blotting. Both isoforms were expressed in all tissues tested but at different levels. Bacterially expressed CKI alpha isoforms were active and therefore biochemically characterized. CKI alpha and CKI alpha L proteins were demonstrated to have casein kinase I catalytic properties. More importantly, the recombinant isoform proteins exhibited differences in binding and activity toward common CKI substrates. These observations demonstrate that the alpha L insert within the kinase domain modulates substrate kinetics. These kinetic differences suggest that CKI alpha and CKI alpha L may perform different biological roles.

Differential expression of DNA polymerase alpha in normal and transformed human fibroblasts.

The expression of DNA polymerase alpha (pol alpha) was studied in human fibroblast lines W138 (fetal lung) and GM3529 (skin, established from a 66 yr old donor), and their Simian virus 40 (SV40) large tumor antigen (TAg)-transformed corollaries, 2RA and 2-1 respectively. Both SV40-transformed and pSV3.neo (SV40-derived plasmid)-transformed cells express TAg, a virally encoded protein not expressed by the normal parent cell lines. Northern blot hybridization studies showed increased recovery of pol alpha mRNA from transformed cells compared with normal cells. This increase was correlated with increased pol alpha mRNA transcription as determined by nuclear run-on assays. Northern blot analyses also showed an increase in the instability of translationally active pol alpha mRNA in transformed cells. The results suggest that TAg, in addition to its dsDNA binding, pol alpha binding, retinoblastoma protein binding and helicase activities, may be involved either directly or indirectly in regulation of the steady state mRNA levels of pol alpha at the transcriptional level in both fetal and aged donor-derived transformed fibroblasts.

Parallel computation of genetic likelihoods using CRI-MAP, PVM, and a network of distributed workstations.

We have developed a version of the CRI-MAP computer program for genetic likelihood computations that runs the FLIPS and ALL functions of CRI-MAP in parallel on a distributed network of workstations. The performance of CRI-MAP-PVM was assessed in several linkage analyses using the FLIPS option of CRI-MAP on a map of 85 microsatellite markers for human chromosome 1. These analyses showed excellent speedup and efficiency and low distribution overhead. In addition, we have adapted the MultiMap program for automated construction of linkage maps to use CRI-MAP-PVM. These improvements significantly reduce the time required to compare likelihoods of different marker orders. Thus, the construction of linkage maps can proceed in a more timely fashion, in keeping with recent advances in genotyping technology.

Age-related changes in expression and activity of DNA polymerase alpha: some effects of dietary restriction.

DNA polymerase alpha (pol alpha) purified from human diploid fibroblasts (HDF) and from livers of C57BL/6N mice showed age-related decreases in: (1) mRNA levels; (2) the amount of enzyme isolated per cell; and (3) enzyme activity (HDF); as well as: a) the amount of enzyme isolated; b) the specific activity; and c) the enzyme fidelity (liver). Hepatic pol alpha from dietary restricted (DR) mice exhibited less of a decline in specific activity and copied synthetic DNA templates with relatively higher fidelity than did enzymes from animals fed ad libitum (AL). Pol alpha from fetal-derived HDF exhibited increased expression compared with aged donor-derived HDF, with both fetal and old cell pol alpha in normal cells being expressed at lower levels than in their transformed cell corollaries. Treatment of human pol alpha from aged donor-derived HDF with a pol alpha accessory protein isolated from log phase murine cells resulted in increased pol alpha binding of DNA and increased pol alpha activity. However, highly active pol alpha isolated from fetal-derived or transformed HDF, or from transformed murine cells, showed little or no activity enhancement in the presence of accessory protein. These data indicate that, as a function of increased age, there is a decrease in pol alpha expression and specific activity in HDF, as well as decreases in specific activity and fidelity of pol alpha in essentially amitotic murine hepatic tissues. Dietary restriction impedes the age-related declines in both activity and fidelity of hepatic pol alpha in mice. The data further indicate that transformation of slowly dividing HDF is associated with increased expression of pol alpha, but suggest that increased expression alone is not sufficient to explain the difference in polymerase activity levels between parental and transformed HDF. Lastly, the data suggest that interaction of pol alpha with an essential accessory protein may be altered as a function of age, an alteration that appears to be correlated with the decline in pol alpha DNA binding and specific activity.

A comparison of DNA polymerase alpha from untransformed and SV40-transformed human fibroblasts.

1. DNA polymerase alpha (pol alpha) isolated from Simian virus 40 (SV40)-transformed cells showed more than 3-fold higher specific activity than pol alpha from normal cells. The enzymes from untransformed and transformed cells also differed in molecular size, thermolability, sensitivity to inhibitors and specificity of template-primer utilization. 2. Western analysis using anti-Tag to probe both a crude cell homogenate and partially purified pol alpha from SV40 transformed cells showed multiple immunoreactive bands with different molecular sizes. 3. While alpha polymerases from both normal and transformed cells exhibited tightly associated primase activity, they showed different DNA binding affinities. 4. These data suggest that T antigen binding to pol alpha alters the initiation of DNA replication and/or the function of pol alpha in SV40-transformed cells, and that pol alpha from SV40-transformed human fibroblasts have different catalytic subunit characteristics than pol alpha from untransformed cells.

Characterization of DNA polymerase alpha from untransformed and pSV3.neo-transformed human fibroblasts.

1. The specific activity of DNA-polymerase alpha isolated from pSV3.neo-transformed cells was more than 9-fold higher than that of polymerase alpha from untransformed cells. 2. Western blot analysis, using anti-SV40 large T antigen, of both a crude cellular extract and of partially purified polymerase alpha from pSV3.neo-transformed cells revealed a single 76 kDa immunoreactive band not found in either crude extracts or partially purified enzyme from untransformed cells. 3. The alpha polymerases from untransformed and transformed cells differed in molecular size, sensitivity to various inhibitors, specificity of template-primer utilization, and binding affinity for DNA cellulose, but showed essentially no differences in Km or Vmax. 4. These data suggest that polymerase alpha isolated from pSV3.neo-transformed cells exhibits altered physical and catalytic characteristics compared with its untransformed cell counterpart, and that those alterations may be associated with increased replication of the genome in plasmid-transformed cells.