Detection of cancer cells in peripheral blood stem cells of women with breast cancer by RT-PCR and cell culture.

The benefit of high-dose therapy and blood stem cell reinfusion for women with high-risk breast cancer is currently under investigation. Contaminations of autologous blood stem cells with cancer cells have been described. Cancer micrometastases may be detected by immunocytochemistry, culture techniques and cytokeratin-19 mRNA reverse transcriptase PCR. Women with breast cancer received adjuvant HD-CTM with peripheral blood stem cell (PBSC) support after surgical therapy and 4 cycles conventional chemotherapy. Peripheral blood stem cells were mobilised by G-CsF and harvested after the third or fourth cycle of standard therapy. Aliquots of PBSC-collections (10(7)-2*10(7) cells) were subjected to CK19-mRNA reverse transcriptase PCR. RNA was extracted by standard methods and reverse transcription was performed with MMV-RT. Integrity of RNA was checked by coamplification of housekeeping sequences. Aliquots of the RT-mix were subjected to PCR-amplification with outer and inner primer pairs, subsequently. A second aliquot of 2*10(7) cells was cultured over 42 days in liquid culture. Cytospins were prepared weekly from cultured cells and evaluated by light microscopy with or without prior immunocytochemistry. Ten leukaphereses from 6 women were available for PCR-analysis and cell culture. Six leukaphereses were negative for CK19-mRNA and for detection of cancer cells by culture technique, two samples were positive for CK19-mRNA and culturally enriched cells and two samples were positive for CK19-mRNA and negative for cultured cancer cells. No sample was positive for cultured cells and negative for CK19-mRNA. Overall, the results corresponded in 80%. Two sensitive techniques for the detection of cancer micrometastases were applied to aliquots from 10 leukaphereses of six breast cancer patients with corresponding results in 80%. PCR-mediated detection of cancer cells was confirmed by culture technique and light microscopy, however, further comparison of CK19-PCR with standard techniques like cell culture and immunocytochemistry is still necessary.

Increased expression of F1ATP synthase subunits in yeast strains carrying point mutations which destabilize the beta subunit.

In yeast strains (S. cerevisiae) carrying a point mutation of the ATP2 gene, which destabilizes the beta subunit of F1 ATP synthase in vitro, the growth rate was reduced significantly, demonstrating that the mutation is also deleterious in vivo. Immunoblots showed that levels of the mutated beta, but also of the wild-type alpha subunit were increased in the mutated strains, together with levels of the corresponding mRNAs (approximately 1.6-fold). Northern analysis showed that this was due to both the appearance of new transcript species as well as upregulation of the cognate transcripts, strongly indicating that the increase was probably due to activation of transcription. Levels of other mitochondrial proteins, e.g. cytochrome c oxidase, were unaffected. We conclude that a specific signal communicates the actual performance of the ATP synthase inside the mitochondria to the nuclear genes encoding its subunits.

Development of thyroid gland volume during the first 3 months of life in breast-fed versus iodine-supplemented and iodine-free formula-fed infants.

The spontaneous development of thyroid gland volume (TGV) during the first 3 months of life was studied in entirely breast-fed infants (n = 21) and compared to those fed an iodine-supplemented formula (n = 19), an iodine-free formula (n = 5), or partially breast-fed in addition to an iodine-free (n = 4) or an iodine-supplemented formula (n = 16). The TGV of the infants and their mothers was determined sonographically in addition to their urinary iodine concentrations 5-7 days postpartum and 3 months later. In ten additional lactating mothers the breast milk concentrations of thyroid hormones and iodine were determined. It was shown that at 3 months of age an infant consuming about 1000 ml breast milk per day receives about 2 micrograms thyroid hormones and 55 micrograms iodine per day. At the end of their first week of life the infants showed a TGV between 0.28 and 1.5 ml (median 0.61 ml) and a urinary iodine concentration between 0.03 and 16.3 micrograms/dl (median 3.0 micrograms/dl). At 3 months of age the TGV of the breast-fed infants had decreased by a median of 0.24 ml (= -34%; median of percentage changes) whereas those fed a formula without iodine had increased by a median of 0.26 ml (= +50%; median of percentage changes). Those receiving an iodine-supplemented formula showed a TGV reduction of 0.14 ml (= +2%; median of percentage changes). The TGV development of the partially breast-fed infants lay between those being exclusively breast or formula fed.(ABSTRACT TRUNCATED AT 250 WORDS)