Engineering CHO cell growth and recombinant protein productivity by overexpression of miR-7.

The efficient production of recombinant proteins by Chinese Hamster Ovary (CHO) cells in modern bioprocesses is often augmented by the use of proliferation control strategies. The most common method is to shift the culture temperature from 37 °C to 28-33 °C though genetic approaches to achieving the same effect are also of interest. In this work we used qRT-PCR-based expression profiling using TLDA™ cards to identify miRNAs displaying differential expression 24h after temperature-shift (TS) from 37 °C to 31 °C. Six miRNAs were found to be significantly up-regulated (mir-219, mir-518d, mir-126, mir-30e, mir-489 and mir-345) and four down-regulated (mir-7, mir-320, mir-101 and mir-199). Furthermore, qRT-PCR analysis of miR-7 expression over a 6 day batch culture, with and without TS, demonstrated decreased expression over time in both cultures but to a significantly greater extent in cells shifted to a lower culture temperature. Unexpectedly, when miR-7 levels were increased transiently by transfection with miR-7 mimic in CHO-K1 cells, cell proliferation at 37 °C was effectively blocked over a 96 h culture period. On the other hand, transient inhibition of endogenous miR-7 levels using antagonists had no impact on cell growth. The exogenous overexpression of miR-7 also resulted in increased normalised (per cell) production at 37 °C, though the yield was lower than cells grown at reduced temperature. This is the first report demonstrating a functional impact of specific miRNA disregulation on CHO cell behavior in batch culture and provides some evidence of the potential which these molecules may have in terms of engineering targets in CHO production clones. Finally, we report the cloning and sequencing of the hamster-specific cgr-miR-7.

MiRNA-29a regulates the expression of numerous proteins and reduces the invasiveness and proliferation of human carcinoma cell lines.

In this study we have identified a functional role for miR-29a in cancer cell invasion and proliferation. MiRNA expression profiling of human NSCLC cell lines indicated that miR-29a levels were reduced in more invasive cell lines. Exogenous overexpression of miR-29a in both lung and pancreatic cancer cell lines resulted in a significant reduction in the invasion phenotype, as well as in proliferation. 2D DIGE proteomic profiling of cells transfected with pre-miR-29a or anti-miR-29a resulted in the identification of over 100 differentially regulated proteins. The fold change of protein expression was generally modest--in the range 1.2-1.7-fold. Only 14 were predicted computationally to have miR-29a seed sequences in their 3' UTR region. Subsequent studies using siRNA to knock down several candidate proteins from the 2D DIGE experiment identified RAN (a member of the RAS oncogene family) which significantly reduced the invasive capability of a model lung cancer cell line. We conclude that miR-29a has a significant anti-invasive and anti-proliferative effect on lung cancer cells in vitro and functions as an anti-oncomir. This function is likely mediated through the post-transcriptional fine tuning of the cellular levels of several proteins, both directly and indirectly, and in particular we provide some evidence that RAN represents one of these.

Proteomic investigation of taxol and taxotere resistance and invasiveness in a squamous lung carcinoma cell line.

Pulse selections on a chemotherapy naive squamous lung carcinoma cell line, SKMES-1, with clinically relevant concentrations of taxanes (taxol or taxotere) resulted in the development of a stable taxotere-resistant variant, SKMES-1-Taxotere and an unstable taxol-resistant variant, SKMES-1-Taxol. Both variants exhibited increased invasiveness in vitro. The unstable nature of SKMES-1-Taxol facilitated looking at factors involved in loss of taxol resistance and increased invasion. The taxotere and taxol-resistant cell lines were 5.9-fold and 12.5-fold resistant to taxotere and taxol respectively. Alterations in expression of/or point mutations in tubulin, the main target of taxanes, is a major mechanism or resistance. However, alterations in expression of beta tubulin were not consistent in the taxane-selected variants. Cross-resistance to adriamycin, vincristine and etoposide (VP-16) was consistent with overexpression of P-glycoprotein (P-gp). However, P-gp alone is not sufficient to confer the complete multiple drug resistance phenotype as all cell lines exhibited cross-resistance to 5-Fluorouracil (5-FU) and more than one mechanism has been linked to taxane resistance. There was no correlation between the fall of taxol resistance in SKMES-1-Taxol and P-gp expression indicating the loss in resistance to be independent of P-gp expression. Furthermore, resistance to the other drugs was not unstable in SKMES-1-Taxol suggesting some parallel mechanisms of resistance. Two-dimensional electrophoresis coupled with matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify alterations in expression of specific proteins associated with taxane resistance. A large number of differentially regulated proteins were identified in the resistant and invasive variants affecting cellular processes including stress response, protein turnover and cytoskeleton proteins.

Purification and identification of a 7.6-kDa protein in media conditioned by superinvasive cancer cells.

BACKGROUND: Selection of the human drug sensitive and invasive cell line (MDA-MB-435S-F) with the chemotherapeutic agent paclitaxel, resulted in the development of drug resistant cell lines displaying enhanced invasion-related characteristics. MATERIALS AND METHODS: Serum-free conditioned media from the human cancer drug-sensitive and invasive cell line (MDA-MB-435S-F) and its paclitaxel-resistant superinvasive variant (MDA-MB-435S-F/Taxol10p4pSI) were analyzed using Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). RESULTS: A differentially expressed protein was observed at 7.6 kDa, which was 4-fold up-regulated in MDA-MB-435S-F/Taxol10p4pSI. The differentially expressed protein was identified using matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS), as a fragment of bovine transferrin. The transferrin receptor was also found to be overexpressed in the superinvasive cell line. CONCLUSION: Cleavage of serum proteins such as transferrin could provide a valuable source of markers for malignant tumours and could also play a role in aspects of cancer pathogenesis, such as tumour cachexia.

Bromodeoxyuridine increases keratin 19 protein expression at a posttranscriptional level in two human lung tumor cell lines.

Keratins form the largest subfamily of intermediate filament proteins and show strict lineage- and differentiation-associated expression in epithelial cells. Little is known about the mechanisms that control keratin protein synthesis in these cells. We have examined the effect of the differentiation-modulating agent, 5'-bromo-2'-deoxyuridine (BrdU), on keratin 19 (K19) expression in two human lung carcinoma cell lines, DLKP and A549. Treatment of both cell lines with 10 microM BrdU for 7 d induced the expression of K19 protein in keratin-negative DLKP cells, and significantly increased K19 protein expression in A549 cells. K19 messenger ribonucleic acids (mRNAs) were detected by Northern blot and reverse transcriptase-polymerase chain reaction analyses in both cell lines, but no increase in K19 mRNA levels was detected in either cell line, even with treatment with BrdU for up to 21 d. This suggests that K19 protein synthesis is normally blocked at a posttranscriptional level in DLKP cells, and BrdU can somehow reverse this block, resulting in the induction of K19 protein synthesis. Treatment of HL60, a leukemic cell line, with BrdU, resulted in noninduction of K19 protein synthesis, and no K19 mRNA transcripts were detected before and after BrdU treatment, possibly suggesting that BrdU is acting in an epithelial-specific manner to reverse a block in K19 protein synthesis in DLKP keratin-negative lung cancer cells. Therefore, DLKP (and A549) may be useful cellular models to investigate if this represents a regulatory step in early lung development or a mechanism whereby tumor cells possess the ability to down-regulate the expression of a more-differentiated phenotype.

Selection with melphalan or paclitaxel (Taxol) yields variants with different patterns of multidrug resistance, integrin expression and in vitro invasiveness.

A melphalan-resistant variant (Roswell Park Memorial Institute (RPMI)-2650Ml) and a paclitaxel-resistant variant (RPMI-2650Tx) of the drug-sensitive human nasal carcinoma cell line, RPMI-2650, were established. The multidrug resistance (MDR) phenotype in the RPMI-2650Tx appeared to be P-glycoprotein (PgP)-mediated. Overexpression of multidrug resistant protein (MRP) family members was observed in the RPMI-2650Ml cells, which were also much more invasive in vitro than the parental cell line or the paclitaxel-resistant variant. Increased expression of alpha(2), alpha(5), alpha(6), beta(1) and beta(4) integrin subunits, decreased expression of alpha(4) integrin subunit, stronger adhesion to collagen type IV, laminin, fibronectin and matrigel, increased expression of MMP-2 and MMP-9 and significant motility compared with the parental cells were observed, along with a high invasiveness in the RPMI-2650Ml cells. Decreased expression of the alpha(2) integrin subunit, decreased attachment to collagen type IV, absence of cytokeratin 18 expression, no detectable expression of gelatin-degrading proteases and poor motility may be associated with the non-invasiveness of the RPMI-2650Tx variant. These results suggest that melphalan exposure can result in not only a MDR phenotype, but could also make cancer cells more invasive, whereas paclitaxel exposure resulted in MDR without increasing the in vitro invasiveness in the RPMI-2650 cells.

Bromodeoxyuridine induces integrin expression at transcriptional (alpha2 subunit) and post-transcriptional (beta1 subunit) levels, and alters the adhesive properties of two human lung tumour cell lines.

Integrins are a family of transmembrane glycoproteins that participate in a wide range of cellular events including proliferation, apoptosis and differentiation. Little is known about the mechanisms that control integrin subunit expression in epithelial cells, especially during lung cell differentiation. We have examined the effect of the differentiation-modulating agent, 5-bromo-2'-deoxyuridine (BrdU), on integrin expression in 2 human lung carcinoma cell lines, DLKP and A549. Treatment of both DLKP and A549 with 10 microM BrdU for 7 days resulted in increased expression of alpha2 and beta1 integrin subunit protein expression. Northern blot and RT-PCR analyses revealed progressively increasing levels of the alpha2 mRNA transcripts following BrdU treatment up to 21 days in both cell lines. However, no increase in beta1 integrin mRNA levels was observed in either cell, suggesting post-transcriptional regulation by BrdU. Treatment of HL-60, a leukaemic cell line, with BrdU up to 21 days resulted in no change in alpha2 or beta1 integrin subunit levels at either protein or mRNA levels suggesting that the change seen in the lung cell lines may be epithelial cell lineage-specific. BrdU has also been found to alter the adhesive properties of A549 and DLKP cells. Treated cells were found to adhere significantly faster to collagen type IV and laminin compared to untreated cells. The results presented here suggest that DLKP (and A549) may be useful cellular models to investigate the role of the alpha2beta1 integrin in lung epithelial cell differentiation.

Bromodeoxyuridine induces keratin protein synthesis at a posttranscriptional level in human lung tumour cell lines.

Keratin intermediate filaments are formed in epithelial cells in a cell- and tissue-specific manner, but much remains unknown regarding the mechanisms which control the synthesis of these proteins. We examined the effect of the differentiation modulation agent, bromodeoxyuridine (BrdU), on two human keratin-negative (by immunocytochemistry) lung cell lines, DLKP and H82, and showed immunohistochemically that treatment with 10 microM BrdU over 7 days induced K8 and K18 protein synthesis in both lines. Immunoprecipitation and Western blot analyses revealed low levels of K8 and K18 proteins in untreated cell homogenates. These levels increased following treatment with BrdU for 7 days. K8 and K18 mRNAs were detected by Northern blot and reverse transcriptase polymerase chain reaction analyses in both lines before BrdU treatment, but no increase in mRNA levels was observed in either cell line over 21 days of treatment. This suggests, firstly, that keratin synthesis is normally blocked at a posttranscriptional level in DLKP and H82 cells, and secondly, that BrdU can reverse this block. A549 is a human lung cell line which contains K8 and K18 proteins. Treatment with BrdU increased K8 and K18 protein levels in these cells. No corresponding increase in K8 mRNA levels occurred, while an apparent increase in K18 mRNA levels was detected. HL-60 is a leukaemic cell-line of haematopoietic rather than epithelial lineage which contains K8 and K18 mRNA transcripts prior to BrdU treatment, but does not contain keratin proteins. Again, K8 and K18 mRNA levels remained unchanged during BrdU treatment. However, neither K8 nor K18 proteins were detected following treatment, although BrdU is known to alter expression of other genes in HL-60 cells. BrdU thus appears to act at a posttranscriptional level and in an epithelial-specific manner to reverse a block in keratin synthesis in keratin-negative lung cancer cells and increase synthesis in keratin-positive lung cancer cells. This may represent a regulatory step in early lung development or a mechanism whereby tumour cells downregulate expression of a differentiated phenotype.

Human lung carcinoma cell line DLKP contains 3 distinct subpopulations with different growth and attachment properties.

Many human solid tumours, particularly lung tumours, contain different subpopulations, the presence of which can complicate diagnosis and treatment, yet few models exist for in vitro studies. We have found that DLKP, a human lung cell line established from a tumour histologically diagnosed as a 'poorly differentiated squamous carcinoma', contains 3 morphologically distinct populations. Three clones corresponding to these populations were established from the parental DLKP cells. Confirmation that the clones were derived from the parental population was obtained by DNA fingerprinting. The clones were designated M (mesenchymallike), I (intermediate) and SQ (squamous). On prolonged subculture, SQ and M can each interconvert with I, but SQ and M do not interconvert. We investigated the growth patterns of these isolated populations in monolayer culture, soft agar, spinner flasks and serum-free medium. In all but the latter assay, the parental DLKP cells grew faster than each of the clones, indicating some form of physiological co-operation between the clones. The growth of the clones themselves varied under the different assay conditions (DLKP-I showing greatest growth in monolayer, in serum-containing and serum-free media but, surprisingly, being unable to grow in soft agar, unlike the SQ and M clones). Addition of fibronectin permitted growth of DLKP-M and DLKP-SQ in serum-free medium at equivalent rates to those of DLKP and DLKP-I. In some cases, morphological adaptation to specific growth conditions was observed. Variation between the clones was also evident in their respective chromosome numbers (with the M clone being predominantly hyperdiploid and the other clones predominantly hypertetraploid) and in their ability to adhere to extracellular matrix proteins, with DLKP-M showing most rapid attachment. Electrical resistance studies revealed the absence of tight junctions from the parental line and clonal subpopulations. Extensive immunohistological studies showed that neither DLKP nor the clones express cytokeratin or any other epithelial marker examined, but neuroendocrine markers were present. Further analysis of these different clonal populations may help to reveal some of the mechanisms involved in lung tumour development and progression.