Irene Abraham

Transfection of a mutant regulatory subunit gene of cAMP-dependent protein kinase causes increased drug sensitivity and decreased expression of P-glycoprotein.

Wild-type Chinese hamster ovary (CHO) cells were transfected with a DNA clone (MT-REV, site A) carrying a mouse gene for a dominant mutant regulatory subunit (RI) gene of cAMP-dependent protein kinase (PKA) from S49 cells along with a marker for G418 resistance. G418-resistant transfectant clone R-2D1 was resistant to 8-Br-cAMP-induced growth inhibition and morphological changes. The cells also did not phosphorylate a 50-kDa protein after cAMP stimulation and had decreased PKA activity, both characteristics of PKA mutants. Northern blot analysis indicated that clone R-2D1 was actively transcribing the MT-REV (site A)-specific RNA. We also tested clone R-2D1 for sensitivity to certain natural product hydrophobic drugs and found increased sensitivity to several drugs including adriamycin. Hypersensitivity to these drugs has previously been shown by us to be a characteristic of a CHO PKA mutant cell line. Expression of the mutant RI gene is also associated with a decrease in expression of the multidrug resistance associated P-glycoprotein (gp170) mRNA and protein. These results show that the PKA mutant RI gene from S49 cells acts as a dominant mutation to reduce the total PKA activity in the CHO transfectants as it does in mouse S49 cells. This study also confirms that reduced PKA activity modulates the basal multidrug resistance of these cells, apparently by causing decreased expression of the mdr gene at the protein and mRNA level.

Staurosporine reduces P-glycoprotein expression and modulates multidrug resistance.

We have investigated the effect of staurosporine and other kinase inhibitors on the mRNA and protein levels of the P-glycoprotein (P-gp) in multidrug resistant (MDR) cells. Treatment of human MDR KB-V1 cells with staurosporine for 24 h caused up to a 50% decrease in the amount of P-gp mRNA and protein present. Co-treatment of KB-V1 cells with verapamil, a known reversal agent, plus staurosporine, H-9, or K252a resulted in an enhanced sensitization of cells to vinblastine than with verapamil alone. These findings support a role for protein kinases in the control of multidrug resistance through effects on P-gp levels.

Identification and sequence of human PKY, a putative kinase with increased expression in multidrug-resistant cells, with homology to yeast protein kinase Yak1.

We have previously shown that several protein kinases are present in higher activity levels in multidrug resistant cell lines, such as KB-V1. We have now isolated a gene that codes for a putative protein kinase, PKY, of over 130 kDa that is expressed at higher levels in multidrug-resistant cells. RNA from KB-V1 multidrug-resistant cells was reverse-transcribed and amplified by using primers derived from consensus regions of serine threonine kinases and amplified fragments were used to recover overlapping clones from a KB-V1 cDNA library. An open reading frame of 3648 bp of DNA sequence predicting 1215 aa, has been identified. This cDNA hybridizes to a mRNA of about 7 kb which is expressed at high levels in human heart and muscle tissue and overexpressed in drug-resistant KB-V1 and HL60/ADR cells. Because its closest homolog is the yeast serine/threonine kinase, Yak1, we have called this gene PKY. PKY is also related to the protein kinase family that includes Cdks, Gsk-3, and MAPK proline-directed protein kinases. This protein represents the first of its type known in mammals and may be involved in growth control pathways similar to those described for Yak1, as well as possibly playing a role in multidrug resistance.

Single-Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells

Abstract: Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus‐infected Spodoptera frugiperda (SF‐9) insect cell system. Culture supernatants contained 2–3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single‐step affinity chromatography procedure using a high‐affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1–2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transacted into the SF‐9 cells coded for pro‐NGF, the NGF recovered after purification was > 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 ± 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 ± 20 pM and 9.6 ± 1.5 pM for a 3‐day primary response and a 1‐day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 ± 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from sub‐maxillary glands.

V79 Chinese hamster lung cells resistant to the bis-alkylator bizelesin are multidrug-resistant

Bizelesin (U-77779) is a highly potent bis-alkylating antitumor agent that is effective against several tumor systems in vitro and in vivo. V79 cells that were 125-to 250-fold resistant to bizelesin developed after constant exposure to gradually increasing concentrations of the drug. Resistant cells exhibited a multidrug-resistant phenotype and genotype as indicated by cross-resistance to several structurally and functionally unrelated drugs, e. g., colchicine, actinomycin D, and Adriamycin, and overexpression of mdr mRNA. Very low levels of cross-resistance to the alkylating, agents cisplatin and melphalan were seen. Multidrug-resistant mouse leukemia (P388/Adriamycin-resistant) and human (KB/vinblastine-resistant) cells were also resistant to bizelesin. Bizelesin resistance was unstable and decreased when cells were grown in the absence of the drug. Resistant and sensitive cell lines had similar levels of glutathione, and bizelesin cytotoxicity for resistant cells was not markedly affected by treatment with buthionine sulfoximine. Cross-resistance between bizelesin and several of its analogs is reported.

Non-glucocorticoid steroid analogues (21-aminosteroids) sensitize multidrug resistant cells to vinblastine

Several members of a group of compounds developed to treat stroke and trauma of the central nervous system are shown also to reverse multidrug resistance in human KB-V1 cells. The most potent reversal agents studied are 21-aminosteroid derivatives (lazaroids), tirilazad mesylate (tirilazad, U-74006F) and U-74389F. Tirilazad sensitizes resistant human cells (KB-V1) to killing by vinblastine by 66-fold, but does not change the sensitivity of the nonresistant parental line, KB-3-1, to vinblastine. KB-V1 cell membranes have high levels of P-glycoprotein, a protein that acts as an efflux pump and is thought to be the major cause of multidrug resistance in these cells. Tirilazad inhibits the photoaffinity labeling of P-glycoprotein with [3H]azidopine in KB-V1 cells more effectively than does verapamil, a standard reversal agent. In addition, tirilazad causes, the increased accumulation of [3H] vinblastine in multidrug resistant KB-V1 cells. Studies of the resistance reversal potential of related compounds suggest that the complex amine portion of tirilazad is important for its reversal activity, while the steroid portion is less important.

Molecular Genetic Analysis of AMP-dependent Protein Kinase

Genetic evidence has been obtained indicating that the responsiveness of CHO cells to cAMP requires an intact cADepPK system. DNA carrying mutant RI and C subunit genes has been transferred to wild-type cells. Recipient cells carrying the DNA have been detected by selecting for expression of the phenotype for cAMP resistance. We are in the process of cloning a functional mutant RI subunit to use as a moveable genetic element that can confer cAMP resistance on recipient cells. This cloned gene should allow us to test hypotheses concerning the mechanism of cAMP regulation of transcription in mammalian cells.

Amyloid precursor protein, microtubule associated protein tau, and ubiquinated inclusions: Brain and spinal cord pathology of various transgenic mice

Formation of intracellular inclusions 1s a common hallmark of neurodegenerative disease. Specifically, Alzheimer‘s disease (AD) is characterized by the formation of large aggregates of Alzheimer’s precursor protein (APP) and ubiquitin, and separate aggregates of tau protein. Familial amyotrophic lateral sclerosis (FALS) is also characterized by the formation of intracellular inclusions containing the ruperoxide diamutase protein (SOD) and ubiquitin. In order to understand any common mechanisms involved in these pathologies, we have studied the progression of the pathological state in various tranagenic mouse lines. These mouse lines include: mice transgenic for APP, tau. APP mice which are also engineered with the AD risk factor ApoF.4, and the mouse model for FALS which consists of a SOD1 tramgene. We have examined a time course of mlmunoreactivity of relevant cellular markers in brain and in the spinal cord. Specifically, we have followed the distribution of ubiquitm. SODI, APP. and tau. Moreover, we have examined the parallel activation of astrocytes and microglia. The analysis of these data reveals common pathological mechani,ma in the two diseases, as well as an increased understanding of the earliest appearance of the pathological state. A deeper understanding of the progression of the disease and early intervention opportunities may present novel windows for therapeutic intervention.