Down-regulation of a calmodulin-related gene during transformation of human mammary epithelial cells.

Abstract

A human cDNA library obtained from cultured normal mammary epithelial cells (HMECs) was searched by subtractive hybridization for genes whose decrease in expression might be relevant to epithelial transformation. One clone identified by this procedure corresponded to a 1.4-kilobase mRNA, designated NB-1, whose expression was decreased >50-fold in HMECs tumorigenically transformed in vitro after exposure to benzo[alpyrene and Kirsten sarcoma virus. Sequence analysis of NB-1 cDNA revealed an open reading frame with a high degree of homology to almodulin. NB-1 expression could be demonstrated by polymerase chain reaction amplification in normal breast, prostate, cervix, and epidermal tissues. The presence of NB-1 transcripts was variable in primary breast carcinoma tissues and undetectable in tumor-derived cell lines of breast, prostate, or other origins.

Short-term primary culture of epithelial cells derived from human breast tumours

“…In normal breast epithelial cultures, the gene was detectable in 13/17 instances, but not found in either tumour-derived epithelial cultures or in tumour cell lines derived from breast or bone. This concurs with other studies that have shown its expression in cultured normal, but not malignant, mammary epithelial cells (Yaswen et al, 1990;Stampfer and Yaswen, 1993). It has also been suggested that in normal breast, the gene is down-regulated in vitro (Yaswen et al, 1990), which may explain the lack of expression of NB-1 in four normal epithelial cultures.…”

“…This concurs with other studies that have shown its expression in cultured normal, but not malignant, mammary epithelial cells (Yaswen et al, 1990;Stampfer and Yaswen, 1993). It has also been suggested that in normal breast, the gene is down-regulated in vitro (Yaswen et al, 1990), which may explain the lack of expression of NB-1 in four normal epithelial cultures. The sensitivity of the PCR, in theory capable of amplifying message from a single cell, unequivocally demonstrates that our cultures contained tumour and not normal epithelial cells.…”

“…This encodes a calmodulin-like protein of unknown function (Yaswen et al, 1990). In normal breast epithelial cultures, the gene was detectable in 13/17 instances, but not found in either tumour-derived epithelial cultures or in tumour cell lines derived from breast or bone.…”

“…Using a modification of a method based on differential centrifugation followed by culture in selective media (Emerson and Wilkinson, 1990;Speirs et al, 1996a), this report describes our experiences in establishing such cultures over a 3-year period and discusses their benefits over cell lines. The cultures have been characterized by a number of criteria including immunocytochemistry and flow cytometry with a range of primary antibodies, RT-PCR to detect the NB-] gene, a calmodulin-like protein of unknown function that is expressed in cultured normal, but not malignant, mammary epithelial cells (Yaswen et al, 1990;Stampfer and Yaswen, 1993) and the presence of increased reductive activity of the steroid-converting enzyme 17p-hydroxysteroid dehydrogenase type I (17-HSD), which is significantly up-regulated in breast tumours (Vermeulen et al, 1986;Peltoketo et al, 1996). (Speirs et al, 1996b).…”

“…After 24 h media was removed and replaced with OM. Cells were (Yaswen et al, 1990;Stampfer and Yaswen, 1993 …”