Loss of cyclin D2 expression in the majority of breast cancers is associated with promoter hypermethylation.

Cyclin D2 is a member of the D-type cyclins, implicated in cell cycle regulation, differentiation, and malignant transformation. It was noted previously that cyclin D2 is not expressed in the majority of breast cancer cell lines, whereas abundant expression was detected in finite life span human mammary epithelial cells. By reverse transcription-PCR and Western blot analysis, we extended this finding to primary breast carcinomas and show that the majority of these tumors lack expression of cyclin D2 mRNA (18 of 24) and protein (10 of 13). In contrast, both luminal and myoepithelial subpopulations of normal breast tissues expressed cyclin D2. Hypermethylation of the CpG island in the promoter was detected by methylation-specific PCR in nearly half of the breast cancers (49 of 106) and was associated with silencing of cyclin D2 gene expression. Promoter hypermethylation was also detected in ductal carcinoma in situ, suggesting that loss of cyclin D2 expression is an early event in tumorigenesis. Our results suggest that loss of cyclin D2 expression is associated with the evolution of breast cancer.

Wilms' tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor-specific promoter methylation.

We analyzed Wilms' tumor suppressor 1 (WT1) expression and its regulation by promoter methylation in a panel of normal breast epithelial samples and primary carcinomas. Contrary to previous reports, WT1 protein was strongly expressed in primary carcinomas (27 of 31 tumors) but not in normal breast epithelium (1 of 20 samples). Additionally, the WT1 promoter was methylated in 6 of 19 (32%) primary tumors, which nevertheless expressed WT1. The promoter is not methylated in normal epithelium. Thus, although tumor-specific methylation of WT1 is established in primary breast cancer at a low frequency, other transcriptional regulatory mechanisms appear to supercede its effects in these tumors. Our results demonstrate expression of WT1 in mammary neoplasia, and that WT1 may not have a tumor suppressor role in breast cancer.

An isoform of the Wilms' tumor suppressor gene potentiates granulocytic differentiation.

WT1 is expressed in hematopoietic progenitor cells and in acute leukemia, but its role in normal and malignant hematopoiesis has not been clearly defined. Alternative splicing of the WT1 mRNA yields several protein isoforms with distinct DNA binding and transcriptional regulatory activities. In this study, we investigated the effect of the WT1 isoform lacking two alternatively spliced sequences (WT1 (-/-)) in 32D cl3 cells, a murine myeloid progenitor cell line. The expression of WT1 (-/-) accelerated the granulocyte-colony stimulating factor (G-CSF)-mediated differentiation of these cells, as judged by morphology and by the expression of differentiation-associated genes and cell surface antigens. WT1 (-/-) inhibited G1/S progression in G-CSF but not in interleukin-3, potentially accounting for its ability to accelerate differentiation. It is likely that dominant-negative mutants previously reported in leukemia patients participate in leukemogenesis by inhibiting this function of the wild-type protein.

Down-regulation of the desmosomal cadherin desmocollin 3 in human breast cancer.

In previous studies using cDNA microarray analysis, we have identified an expressed sequence tag which is consistently down-regulated in six human breast tumor cell lines. In the current study, we have determined this tag to be part of the mRNA sequence of human desmocollin 3, a member of the cadherin superfamily of proteins and an integral component of desmosomes. Desmosomes are sites of adhesion between adjacent cells in layers of epithelia, as well as in some non-epithelial tissues, and play an important role in the maintenance of tissue structure. Northern analysis, quantitative real-time polymerase chain reaction assay and Western blot analysis showed that desmocollin 3 is present in normal and immortalized human mammary epithelial cells, but consistently exhibits a significant, and often complete, down-regulation in breast cancer cell lines and primary breast tumors, both at the mRNA and protein levels.

High cell density fermentation of recombinant Escherichia coli with computer-controlled optimal growth rate.

In recent years recombinant DNA technology has enabled us to produce various proteins of therapeutic importance with microorganisms. As an appropriate host organism, E. coli plays a dominant role. Yields of E. coli dry cell mass in shaker flask culture range from 1-2 g/L, whereas in fermentors up to 10 g dry cells/L can be achieved. ZIMET and GBF have developed a high cell density fermentation process that produces E. coli (on a glucose/mineral salt medium) up to more than 100 g dry cells/L in a special fed-batch mode. This cultivation strategy prevents oxygen limitation and hence the accumulation of acetate and other metabolic byproducts. The specific growth rate can be adjusted so that product formation reaches its optimum value. An example of the production of alpha1-interferon is presented. The high cell density fermentations were realized in 30- and 450-L Chemap fermentors (ZIMET) and in a three-stage bioreactor scale-up system (72, 300, and 1,500 L) developed in cooperation with GBF and B. Braun Melsungen AG. Multiloop controllers were used to control the process variables.

Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli TG1.

Continuous and fed-batch cultures of recombinant Escherichia coli TG1 were carried out in order to study plasmid stability and recombinant product formation at different specific growth rates. The aprotinin::beta-galactosidase gene (Ap::lacZ) was placed under the control of two different promoter/repressor systems, the PLac/lacI (pPLac8) and the lambda PL/cIts857 (pPL6) system. The chemically (0.5 mM IPTG) induced gene expression exhibited higher product activity and plasmid stability than the thermally (40 degrees C) induced expression. In fed-batch cultivations with the more stable E. coli TG1(pPLac8) a special feeding strategy allowed bacterial growth with a constant growth rate mu for several hours up to high cell densities. The cloned gene product activity was noticeably effected by the specific growth rate and the cell density at the moment of induction. In particular, the enzyme activities passed a pronounced maximum value in dependence of the set growth rate. The results indicate that fed-batch cultivation strategies are well suited to produce recombinant gene products.

Control of microbial activity by flow injection analysis during high cell density cultivation of Escherichia coli.

The application of an automated flow injection analysis (FIA) system for on-line determination of microbial activity, during high cell density cultivations of Escherichia coli is reported. Based on a bioelectrochemical principle, the FIA method used a redox mediator (potassium hexacyanoferrate(III)) to facilitate electron transfer from the microorganisms to an electrochemical detector. Assays were carried out using a new sampling device which provided aseptic operation by use of a valve and chemical sterilisation. No sample dilution or pretreatment was necessary for biomass concentrations up to approx. 40 g l-1. The sample volume was 0.5 ml and the overall analysis time was 5 min. FIA signals were found to correlate well with the oxygen uptake rate (OUR). Changes in metabolic activity due to low substrate levels or high inhibitor concentrations in the cultivation medium became obvious from the FIA signals.

Simple fed-batch technique for high cell density cultivation of Escherichia coli.

A simple fed-batch process for high cell density cultivation of Escherichia coli TG1 was developed. A pre-determined feeding strategy was chosen to maintain carbon-limited growth using a defined medium. Feeding was carried out to increase the cell mass concentration exponentially in the bioreactor controlling biomass accumulation at growth rates which do not cause the formation of acetic acid (mu < mu crit). Cell concentrations of 128 and 148 g per 1 dry cell weight (g l-1 DCW) were obtained using glucose or glycerol as carbon source, respectively.

High cell density cultivation of Escherichia coli at controlled specific growth rate.

A high cell density cultivation (HCDC) for growth of Escherichia coli in an especially designed glucose/mineral salt medium is proposed. The HCDC essentially starts as a batch process which is followed by a two-phase fed-batch cultivation. After unlimited growth at mu max = 0.45 h-1 in the batch part, growth was controlled at a reduced specific growth rate (mu = 0.11 h-1 less than mu max) over a period of 3 doubling times in which the biomass concentration increased from 12 to 95 g 1(-1) (phase 1 of fed-batch cultivation). Control of growth (mu) was realized by a PO2 control loop (by variation of glucose feeding) and a mu control loop (by variation of agitation speed N) while the actual mu was calculated from the off-gas composition. If the agitation rate cannot be increased anymore the mu controller is switched off (end of phase 1). In the following phase 2, mu declines, however, the still acting pO2 (glucose) controller guarantees sufficient O2 supply till the end of the cultivation with a biomass concentration of 110 g 1(-1) (dry mass). The proposed HCDC suppresses generation of inhibitory by-products and the high yield coefficients indicate the economy of the process.

HOXA5 regulates expression of the progesterone receptor.

The majority of breast carcinomas show reduced or no expression of the transcription factor, HOXA5. Recently, we have shown that HOXA5 is a potent transactivator of p53 in breast cells and thus may affect the response of breast cancer cells to DNA damage. To determine whether HOXA5 played a role in growth and homeostasis in breast cells, we studied its interaction with the progesterone receptor. The progesterone receptor (PR) belongs to the superfamily of nuclear receptors whose members co-ordinate morphogenesis of the mammary gland in response to binding to their cognate ligands. An increased expression of the endogenous PR gene was seen in MCF-7 cells following induced expression of an exogenously transfected HOXA5 gene. HOXA5, but not HOXB4, -B5, or -B7 activated the PR promoter in two breast cancer cell lines, MCF-7 and Hs578T. Deletion and mutation analysis of the promoter identified a single HOXA5-binding site required for transactivation of the PR gene by HOXA5. HOXA5 binds directly to this site in the PR promoter. Thus, HOXA5 may behave as a transcriptional regulator of multiple target genes, two among which are p53 and the progesterone receptor.