Level Of Gene Amplification Research Articles

HER-2 gene amplification correlates with higher levels of angiogenesis and lower levels of hypoxia in primary breast tumors.

This study investigated the connection among HER-2 gene amplification, HER-2 protein expression, and markers of tumor angiogenesis and oxygenation in patients with operable, invasive breast tumors. From 1988 to 1995, 425 patients with metastatic breast cancer were enrolled in a study of high-dose chemotherapy with autologous transplant. Primary tumor blocks were obtained and evaluated using immunohistochemistry (IHC) staining of vessels with von Willebrand factor antibody. Mean microvessel densities (MVD) were determined by counting von Willebrand factor stained cells in three separate "vascular hot spots" using image analysis. Tumor samples were also stained for HER-2 by IHC, HER-2 gene amplification by fluorescence in situ hybridization, carbonic anhydrase 9 by IHC, and vascular endothelial growth factor (VEGF) by IHC. Plasma from 36 patients with primary tumor samples had VEGF (R&D Systems, MN) and d-dimer (American Diagnostica, Greenwich, CT) levels determined. There was a significant positive correlation between HER-2 gene amplification and both maximum and average MVD (Spearman coefficient = 0.51 and 0.50; P = 0.03 and 0.05, respectively). There was an inverse correlation with HER-2 gene amplification and expression of the tumor hypoxia marker CA-9 (chi(2) P = 0.02). The level of HER-2 gene amplification correlated with plasma d-dimer levels (Spearman coefficient = 0.43; P = 0.021). Interestingly, tumors with HER-2 by IHC had decreased amounts of VEGF staining (chi(2) = 5.81; P = 0.01). There was no correlation between HER-2 by IHC and MVD or d-dimer. Of all of the variables examined, only average (P = 0.0016) and maximum MVD (P = 0.0128) predicted disease-free survival (Cox univariate model). HER-2-amplified breast cancers have increased amounts of angiogenesis, decreased amounts of hypoxia, and increased markers of fibrin degradation. These findings have prognostic, predictive, and therapeutic implications in breast cancer treatment.

Colcemid resistance in murine SEWA cells: non-Pgy gene amplification at low levels of resistance and preferential Pgy2 gene amplification at high levels of resistance.

Mammalian cell lines often become multidrug-resistant to cytotoxic drugs by amplification and/or overexpression of the P-glycoprotein (Pgy) genes. However, several malignant cell lines seem to acquire low levels of drug resistance by non-P-glycoprotein mediated mechanisms. We report here on cytogenetical signs of non-Pgy gene amplification in murine SEWA cells during the early steps of selection in Colcemid (COL). In line TC13COL0.01, rare cells exhibited a homogeneously staining region (HSR) distally in chromosome 16. As the COL-concentration was raised the HSR-chromosome was retained and, in addition, the cells developed numerous double minutes (DMs). The DMs, but not the HSR, contained amplified Pgy genes. The HSR may correspond to amplified heat shock protein 70 (Hsp70) genes, detected by Southern analysis. A second low-level COL-resistant line, TC13D70.01, contained DMs but showed no amplification of Pgy, Hsp70, Hsp90, alpha- or beta-tubulin genes. In higher COL-concentration, P-glycoprotein mediated drug resistance was induced. In contrast to actinomycin D-resistant SEWA cells, in which higher amplification levels of Pgy1 than of Pgy2 are regularly present, the COL-resistant lines showed a preference for Pgy2 gene amplification. These results are in line with the suggestion that the murine Pgy1 and Pgy2 genes have overlapping but distinct drug specificities.

Amplification of a 280-Kilobase Core Region at the ERBB2 Locus Leads to Activation of Two Hypothetical Proteins in Breast Cancer

Amplification of the ERBB2 oncogene at 17q12 is clinically the most relevant genetic aberration in breast cancer and several studies have linked ERBB2 activation to poor clinical outcome. The development of targeted antibody-based therapy for ERBB2-overexpressing tumors and the possible role of ERBB2 as a predictor of chemotherapy treatment response have further emphasized the essential role of ERBB2 in breast cancer. Here, we performed a detailed characterization of the molecular events occurring at the ERBB2 amplicon in primary breast tumors. Analysis of the amplicon structure in 330 breast tumors by fluorescence in situ hybridization to a tissue microarray revealed a 280-kb common region of amplification that contains 10 transcribed sequences, including eight known genes. The expression levels of these 10 transcripts were determined in 36 frozen samples of grade-matched ERBB2-amplified and -nonamplified (as determined by fluorescence in situ hybridization) primary breast tumors by using quantitativereal-time reverse transcriptase-polymerase chain reaction. A highly significant association between amplification and expression levels was observed for six of these genes, including ERBB2 and two uncharacterized hypothetical proteins, MGC9753 and MGC14832. These results support the recent findings on the influence of copy number on gene expression levels and highlight novel genes that might contribute to the clinical behavior of ERBB2-amplified breast tumors.

HER2 and COX2 expression in human prostate cancer

COX2 and HER2 expression are associated with a poor prognosis in prostate cancer and HER2 has been linked to COX2 expression in colorectal cancer. The association between COX2 and HER2 expression was investigated in 117 patients with prostate cancer (89) or Benign prostatic hyperplasia (BPH) (28). Tissue was analysed for HER2 amplification by fluorescent in situ hybridisation, and HER2 and COX2 protein expression by immunohistochemistry (IHC). All tumours analysed expressed COX2 at a significantly higher level than BPH tissue (P=0.041). Only low levels of HER2 gene amplification (8%, 7/89) and HER2 protein expression (12%, 11/89) were observed. HER2 protein expression was rarely observed and did not correlate with HER2 amplification or COX2 expression. Although HER2 does not drive COX2 expression in prostate cancer, this study identified high levels of COX2 expressed in locally advanced prostate cancer, suggesting COX2 could be a potential therapeutic target. COX2 inhibitors are currently being used in clinical trials for the treatment of other tumour types.

Array rank order regression analysis for the detection of gene copy-number changes in human cancer

cDNA microarray technology has been applied to the detection of DNA copy-number changes in malignant tumors. Test and control genomic DNA samples are differentially labeled and cohybridized to a spotted cDNA microarray. The ratio of test to control fluorescence intensities for each spot reflects relative gene copy number. The low signal-to-noise ratios of this assay and the variable levels of gene amplification and deletion among tumors hamper the detection of deviations from the diploid complement. We describe a regression-based statistical method to test for altered copy number on each gene and apply the technique to copy-number profiles in 10 thyroid tumors. We show that a novel transformation of fluorescence ratios into array rank order efficiently normalizes the heterogeneity among copy-number profiles and improves the reproducibility of the results. Array rank order regression analysis enhances the detection of consistent changes in gene copy number in solid tumors by cDNA microarray-based comparative genome hybridization.

Mechanism of action of anti-HER2 monoclonal antibodies: scientific update on trastuzumab and 2C4.

The HER family of transmembrane tyrosine kinase receptors is composed of four members, BER1 to HER4. HER2 is a ligand-orphan receptor expressed in many human tumors and overexpressed in 25-30% of breast cancers. HER2 amplifies the signal provided by other receptors of the HER family by forming heterodimers. The essential role of HER2 in the HER signaling network led to the development of anti-HER2 monoclonal antibodies (MAbs) for cancer therapy. In particular, the humanized MAb trastuzumab (Herceptin) has antitumor activity against HER2-overexpressing human breast tumor cells and is widely used for the treatment of women with HER2 overexpressing breast cancers. Trastuzumab induces HER2 receptor downmodulation and, as a result, inhibits critical signalling pathways (i.e. ras-Raf-MAPK and PI3K/Akt) and blocks cell cycle progression by inducing the formation of p27/Cdk2 complexes. Trastuzumab also inhibits HER2 cleavage, preceding antibody-induced receptor downmodulation, and this effect might contribute to its antitumor activity in some cancers. In vivo, trastuzumab inhibits angiogenesis and induces antibody-dependent cellular cytotoxicity. A limitation of trastuzumab is that its activity is largely restricted to breast cancers with the highest level of HER2 overexpression or HER2 gene amplification. However, there is a large population of breast cancers and of many other tumors that have low or moderate HER2 expression. In such tumors, HER2 functions as a preferred coreceptor to form heterodimers with HER1 (EGFR), HER3 or HER4. For this reason, a humanized monoclonal antibody, called 2C4, that targets the role of HER2 as a coreceptor is under active development. 2C4 binds to a different epitope of HER2 ectodomain than trastuzumab and sterically hinders HER2 recruitment in heterodimers with other HER receptors. This results in the inhibition of signalling by HER2-based heterodimers both in cells with low and high HER2 expression. In vitro and in vivo antitumor activity has been reported in a range of breast and prostate tumor models. Therefore, 2C4 may have potential against a wide variety of solid tumors. Phase I trials are underway.

Is Fluorescence In Situ Hybridization Really Superior to HercepTest?

Is Fluorescence In Situ Hybridization Really Superior to HercepTest?

Frequent cyclin D1 gene amplification and protein overexpression in oral epithelial dysplasias.

Amplification of the cyclin D1 gene has been identified in 17-55% of head and neck squamous cell carcinoma. In some tumors, this alteration has been associated with decreased survival and increased recurrence rates. In precancerous lesions of the mouth, the frequency of cyclin D1 gene amplification is not known. In addition, it is unknown whether amplification of the gene translates to overexpressed cyclin D1 protein in these lesions. We examined 59 formalin-fixed, paraffin embedded tissue biopsies of oral epithelial dysplasias (OED) and 25 oral squamous cell carcinoma (SCC) from the floor of the mouth for cyclin D1 gene and protein levels. Genomic DNA was extracted from laser microdissected lesional tissue and a duplex, quantitative PCR assay was used to determine the amplification of the cyclin D1 gene relative to interferon-gamma. Cyclin D1 protein expression was determined using immunohistochemistry and counting positive nuclei by computer image analysis. We found cyclin D1 gene amplification in 41% of mild, 45% of moderate and 24% of severe OEDs. Cyclin D1 was amplified in 36% of SCC. Overexpression of cyclin D1 protein was identified in 29% of mild, 47% of moderate, 29% of severe OED's, and in 32% of SCC. Overexpression of cyclin D1 protein was identified in similar proportions of all grades of dysplasia and SCC. There were statistically significant correlations identified between gene and protein levels in all categories of disease. We concluded that amplification of the cyclin D1 gene is frequent in OED and that duplex, quantitative polymerase chain reaction is a reliable method to detect this change in routinely processed biopsies. The strong correlation between cyclin D1 gene amplification and protein levels suggests that this method may be suitable to assess cyclin D1 gene status in tissues not suitable for protein analysis.

Gene amplification and expression of the steroid receptor coactivator SRC3 (AIB1) in sporadic breast and endometrial carcinomas.

Steroid receptor coactivator 3 (SRC3) functions as a coactivator for nuclear receptor mediated transcriptional activation. It binds to nuclear receptors in a ligand-dependent fashion and recruits other factors such as CBP and p300 to the transactivation complex. Due to its function as activator of nuclear receptors, overexpression of SRC3 might enhance their effects. Gene amplification is a common mechanism that causes overexpression, already described for oncogenes like c-erbB2, c-myc and int2. In this study, SRC3 gene amplification and expression levels were analyzed in 127 sporadic breast carcinomas, 30 endometrial carcinomas and different cell lines (MCF7, HeLa, Ishikawa, T47D, BT-20, SK-BR-3, HEC-1a, RL 95-2, OVCAR3 and A-431). To determine gene amplification and mRNA expression levels, quantitative differential PCR and RT-PCR were performed in combination with fluorescent DNA technology. Gene amplification was not found in any of the breast and endometrial carcinomas, but was found in the carcinoma cell lines MCF7 (10-fold) and HeLa (3-fold). SRC3 overexpression was detected in 13% (3/23) of breast carcinomas and 17% (5/30) of endometrial carcinomas, as well as in MCF7 and HeLa cells. Thus, SRC3 overexpression found in breast and endometrial tumors is not caused by SRC3 gene amplification. A carcinogenic potential provided by SRC3 overexpression has to be elucidated in further studies.

A one-step gene amplification system for use in cultured mammalian cells and transgenic animals.

Gene amplification is widely used for the production of pharmaceuticals and therapeutics in situations where a mammalian system is essential to synthesise a fully active product. Current gene amplification systems require multiple rounds of selection, often with high concentrations of toxic chemicals, to achieve the highest levels of gene amplification. The use of these systems has not been demonstrated in specialised mammalian cells, such as embryonic-stem cells, which can be used to generate transgenic animals. Thus, it has not yet proved possible to produce transgenic animals containing amplified copies of a gene of interest, with the potential to synthesise large amounts of a valuable gene product. We have developed a new amplification system, based around vectors encoding a partially disabled hypoxanthine phosphoribosyltransferase (HPRT) minigene, which can achieve greater than 1000-fold amplification of HPRT and the human growth hormone gene in a single step in Chinese hamster-lung cells. The amplification system also works in mouse embryonic-stem cells and we have used it to produce mice which express 30-fold higher levels of human protein C in milk than obtained with conventional transgenesis using the same protein C construct. This system should also be applicable to large animal transgenics produced by nuclear transfer from cultured cell lines.

The rate of folate receptor alpha (FR alpha) synthesis in folate depleted CHL cells is regulated by a translational mechanism sensitive to media folate levels, while stable overexpression of its mRNA is mediated by gene amplification and an increase in transcript half-life.

DC-3F/FA3 cells (FA3) were obtained by selection of Chinese hamster lung fibroblasts for growth in folic acid free media, supplemented with 15 pM [6S]-5-formyltetrahydrofolic acid. These cells, as a result of low level gene amplification and RNA stabilization, were found to overexpress folate receptor alpha (FR alpha) mRNA by more than five hundred fold. The expression level of the receptor, a 43 kDa GPI-linked plasma membrane glycoprotein, was found to be inversely related to changes in media folate concentrations while its steady state mRNA level remained unaffected. In low folate, the rate of receptor synthesis was found to increase by more than three fold, while its half-life stabilized as compared to that observed in high folate media. Although DC-3F cells were found to contain low amounts of FR alpha mRNA, receptor expression was undetectable, and changing media folate concentrations had no effect on the expression of either. Hence, while selection for growth in low folate leads to stable overexpression of FR alpha mRNA, receptor expression is regulated at the level of protein synthesis by a mechanism sensitive to media folate levels.

Quantitative variation and selection of esterase gene amplification in Culex pipiens.

Although descriptions of evolutionary mechanisms are common in the literature, very few studies focus on the possible evolution of the adaptive genes themselves, i.e. their quantitative and qualitative changes. Evolution of insecticide resistance in Culex pipiens is a suitable model for studying such processes. In this species, organophosphorous insecticide resistance can be achieved through the overproduction of esterases that sequester the insecticide, and this overproduction can be caused by gene amplification. It is generally assumed, but never verified, that esterase activity, and therefore resistance, is monotonically related to gene amplification. We have analysed resistance, esterase activity and gene amplification in different laboratory strains and natural populations in order to detect variability and to infer effects of selection on these factors. We have shown that resistance, esterase activity and amplification covary, that insecticide selection is able to increase amplification levels, and that a fitness cost is probably attached to the amplification in laboratory strains, related to the level of amplification. The importance of variation in gene amplification level is discussed and some evolutionary implications are proposed.

Reduction ofmdrlGene Amplification in Human Multidrug-Resistant LoVo DX Cell Line Is Promoted by Triple Helix-Forming Oligonucleotides

We have demonstrated previously that the GT triplex-forming oligodeoxyribonucleotide (TFO) d(TGTGTTTTTGTTTTGTTGGTTTTGTTT), named TFO ID, targeted to a polypyrimidine-polypurine coding sequence located within human multidrug-resistance mdrl gene, specifically and significantly reduced mdrl mRNA levels in the drug-resistant T-leukemic CEM-VLB100 cell line. In this article, we demonstrate that TFO 1D is effective at inhibiting not only transcription but also replication of mdrl genes, leading to a loss of amplified gene copies in the drug-resistant colon adenocarcinoma LoVo DX cell line. In contrast, TFO ID does not alter replication of the constitutive mdrl gene copy in the corresponding parental sensitive LoVo 109 cell line. A specific reduction in mdrl gene amplification levels was also obtained with the pyrimidine TFO d(CTTTTTCTTTTCTTCCTTTTCTTT), named TFO 24TC, directed against the same polypyrimidine-polypurine sequence of the mdrl gene. We suggest that triple helix-forming oligonucleotides might affect the replication of unstable chromosomal elements as amplicons in actively replicating cells by causing a local impairment of DNA polymerase activity. This study lends support to the notion that TFO may be used to reduce gene amplification aiming to control neoplastic progression in cancer cells bearing amplified oncogenes.

Quantitative analysis of transcription and translation in gene amplified Chinese hamster ovary cells on the basis of a kinetic model.

The elevation of expression levels for secreted glycoproteins by gene amplification in mammalian cells shows a saturation behavior at high levels of gene amplification. At high expression levels a drop in the secretion efficiency for the recombinant protein occurs (Schröder and Friedl, 1997), coinciding with the appearance of misfolded protein in the cell. In this communication we investigated whether additional limitations exist at the levels of transcription and translation. Four Chinese hamster ovary (CHO) cell lines expressing different amounts of human antithrombin III (ATIII) were used as a model system. A tenfold increase in the ATIII cDNA copy number from the lowest to the highest producing cell line coincided with a 38-fold increase in ATIII mRNA levels, and an 80-fold increase in the amount of intracellular ATIII levels. The data was analyzed using a simple kinetic model. The following conclusions were derived: I. The transcriptional activity for the recombinant protein is not saturated. II. Translation itself is not saturated either, but may be downregulated as secretion efficiency drops. III. Two explanations for the previously reported drop in secretion efficiency for the recombinant protein with increasing expression level are possible: A. Protein degradation is an alternative fate for translated ATIII and the fraction of ATIII degraded after translation increases as expression level is increased. B. Translation is downregulated as the secretory apparatus becomes exhausted to maintain cell viability.

Cell proliferation rate by MIB-1 immunohistochemistry predicts postradiation recurrence in prostatic adenocarcinomas.

We examined whether the cell proliferation index by MIB-1, HER-2/neu gene amplification, Gleason grade, and pretreatment level of serum prostate specific antigen (PSA) correlated with postradiation recurrence (PRR) in patients with prostatic adenocarcinoma. Formalin-fixed, paraffin-embedded tissue sections from 42 pretreated cases of prostatic adenocarcinoma (38 needle biopsy and 4 transurethral resection specimens) were immunostained for MIB-1 (MMI, Ventana Medical Systems, Tucson, Ariz). HER-2/neu gene amplification was analyzed by fluorescence in situ hybridization using the Oncor unique sequence probe (Oncor, Gaithersburg, Md). The cell proliferation index by MIB-1 was determined by labeling index; levels of HER-2/neu were analyzed semiquantitatively. Twenty-three of 42 patients (55%) were considered to have PRR on the basis of consecutive elevations of serum levels of PSA to greater than 1.5 ng/mL after completion of treatment (mean follow-up time, 33.4 months). The cell proliferation index correlated with PRR on both univariate and multivariate analyses. Of the 23 tumors that showed PRR, 18 (78%) revealed a high cell proliferation index, compared with 6 of 19 cases (32%) that showed no PRR. Twelve of 23 cases of prostatic adenocarcinoma (52%) in the recurrent group showed HER-2/neu gene amplification, compared with 5 of 19 (26%) in the nonrecurrent group; these findings reached near significance on univariate analysis. Pretreatment levels of serum PSA also reached significance on multivariate analysis. In this preliminary study, the cell proliferation index by MIB-1 reached independent significance in predicting PRR in patients with prostatic adenocarcinoma, whereas HER-2/neu amplification by fluorescence in situ hybridization reached near significance.

Expression of the c-Myc protein in childhood medulloblastoma.

The purpose of this study was to determine the incidence of c-Myc protein expression in medulloblastoma/primitive neuroectodermal tumor (MB/PNET) and to identify mechanisms in addition to c-myc gene amplification that lead to increased protein expression. We analyzed c-myc gene copy number, mRNA level and protein expression in a panel of MB/PNET cell lines. C-Myc protein levels were assessed in tumor specimens and cell lines using immunohistochemical staining with a c-Myc-specific monoclonal antibody. Southern analysis confirmed c-myc gene amplification in the D425 MED cell line and re-arrangement of one allele in D283 MED, which was analyzed further and appeared to represent a small deletion 3' of exon 3. C-myc transcript levels were dramatically elevated in both lines. Using a c-myc probe, fluorescence in situ hybridization (FISH) showed c-myc present in 3 tandem copies at 8q24 in D283 MED and multiple copies as double minutes in D425 MED. Immunohistochemistry showed c-Myc protein expression in 9 of 10 tumors and all cell lines, regardless of gene amplification status or level of mRNA expression. c-Myc protein expression is common in MB/PNET tumor specimens and cell lines. Elevated protein levels are observed in the absence of amplification, suggesting that multiple mechanisms of c-myc dysregulation may be involved in MB/PNET. These studies support a role for c-Myc in the development of this common childhood tumor.

Unbalanced growth in mouse cells with amplified dhfr genes

When grown in the absence of methotrexate, cells carrying unstably amplified dihydrofolate reductase (dhfr) genes have a growth disadvantage that is a function of their level of gene amplification. Although this growth disadvantage is thought to drive the loss of unstably amplified dhfr genes in the absence of methotrexate, its mechanism is not understood. The present studies of murine cell lines with different levels of dhfr gene amplification demonstrate that such cells experience increased unbalanced growth (excess RNA and protein content relative to DNA content) with increased levels of dhfr gene amplification. Stathmokinetic analysis of a cell line with unstably amplified dhfr genes showed that the unbalanced growth was associated with a very low rate of G1/S transit, which suggests that amplified DNA sequences may activate a cell cycle checkpoint at the G1/S boundary. Hydroxyurea, which is known to induce rapid elimination of amplified genes at sub-cytotoxic concentrations, also inhibits the cell cycle at the G1/S transition and causes unbalanced growth. Earlier work has shown that hydroxyurea selectively targets those cells within the heterogeneous drug resistant cell populations which have the highest amplified gene dosage. The finding that unstable gene amplification and hydroxyurea have similar effects on the cell suggests that hydroxyurea may achieve this selective targeting by pushing those cells with the highest levels of gene amplification over a critical stress threshold to cause growth arrest or cell death.

Unbalanced growth in mouse cells with amplified dhfr genes

AbstractWhen grown in the absence of methotrexate, cells carrying unstably amplified dihydrofolate reductase (dhfr) genes have a growth disadvantage that is a function of their level of gene amplification. Although this growth disadvantage is thought to drive the loss of unstably amplified dhfr genes in the absence of methotrexate, its mechanism is not understood. The present studies of murine cell lines with different levels of dhfr gene amplification demonstrate that such cells experience increased unbalanced growth (excess RNA and protein content relative to DNA content) with increased levels of dhfr gene amplification. Stathmokinetic analysis of a cell line with unstably amplified dhfr genes showed that the unbalanced growth was associated with a very low rate of G1/S transit, which suggests that amplified DNA sequences may activate a cell cycle checkpoint at the G1/S boundary. Hydroxyurea, which is known to induce rapid elimination of amplified genes at sub‐cytotoxic concentrations, also inhibits the cell cycle at the G1/S transition and causes unbalanced growth. Earlier work has shown that hydroxyurea selectively targets those cells within the heterogeneous drug resistant cell populations which have the highest amplified gene dosage. The finding that unstable gene amplification and hydroxyurea have similar effects on the cell suggests that hydroxyurea may achieve this selective targeting by pushing those cells with the highest levels of gene amplification over a critical stress threshold to cause growth arrest or cell death.

Quantitative analysis of cyclin D1 messenger RNA expression in head and neck squamous cell carcinomas.

Cyclin Dl is thought to play a critical role in the G1/S phase transition of the cell cycle. Amplification of this gene has been reported in several types of human neoplasms including breast, lung, esophageal, and head and neck tumors. In this study, we have analyzed the relative level of expression of cyclin D1 messenger RNA (mRNA) in fresh specimens of head and neck squamous cell carcinoma (HNSCC), and investigated the concordance of the overexpression of cyclin D1 mRNA with gene amplification. Levels of cyclin D1 mRNA were analyzed hy a modified method of competitive reverse transcription‐polymerase chain reaction and levels of cyclin D1 gene amplification were evaluated by Southern blot hybridization in a series of 23 matched normal mucosas and HNSCC. Overexpression of cyclin D1 mRNA was observed in 10 of 23 cases (43.5%) of HNSCC, ranging from 2 to 50‐fold higher than the normal control. Twelve of 23 cases could be evaluated by Southern blot hybridization, and gene amplification was found in only 2 of 12 cases (16.7%), These findings suggest that cyclin D1 plays an important role in tumorigenesis of HNSCC, and gene amplification is not one of the major mechanisms for overexpression of cyclin D1.

Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein

Overexpression of recombinant proteins in animal cells is commonly achieved by using gene amplification techniques. Gene amplified cells possess up to several thousand genes coding for the target protein. Constitutive expression of these genes leads to high levels of the corresponding mRNA species and the immature protein in the cell. Inefficient processing of these precursors may result from their great abundance in the cell. To study the influence of elevated intracellular levels of a recombinant protein on its maturation and secretion, we examined the maturation and secretion of human antithrombin III (hATIII) in Chinese hamster ovary (CHO) cells at different levels of gene amplification. No loss of vitality was caused by elevated secretion of hATIII. As the intracellular hATIII content increased, the efficiency of hATIII secretion decreased steadily. The state of intracellular hATIII from the different cell lines was studied by determining the specific heparin cofactor activity of hATIII. Intracellular hATIII from the highest amplified cell line displayed a lowered specific heparin cofactor activity indicating the presence of malfolded, only partially folded, or incompletely or incorrectly posttranslationally modified hATIII in this cell line. Thus, the ability of CHO cells to fold and/or introduce posttranslational modifications and subsequently to secrete the recombinant protein becomes saturated, and therefore these processes may become limiting for protein secretion at highly elevated expression levels. This limitation was not due to a general exhaustion of the secretory capacity of the cells because hATIII constituted only a minor fraction of the secreted proteins, even at high expression levels.