Analysis of the mechanisms that maintain coordinate production of $\alpha$- and $\beta$-tubulin in mammalian cells

Alpha and beta tubulin are essential proteins in all eukaryotic cells. To study how cells maintain coordinate levels of these two interacting proteins, we have used PCR to add a 9 amino acid epitope from influenza hemagglutinin protein onto the carboxyl terminus of $\alpha$1 and $\beta$1-tubulin. The chimeric tubulin genes (HA$\alpha$1 and HA$\beta$1) were transfected into CHO cells and cell lines that stably express each gene were selected. Cells transfected with HA-tubulin do not exhibit any gross changes in growth or morphology. Immunofluorescence analysis demonstrated that HA-tubulins incorporate into both cytoplasmic and spindle microtubules. A quantitative biochemical assay was used to show that HA-tubulins incorporate into microtubules to a normal extent and do not alter the steady state distribution of endogenous tubulin between monomer and polymer pools. Two-dimensional gel analysis of pulse-labeled cells indicated that when HA$\beta$1-tubulin is expressed at high levels, it slightly represses the synthesis of the endogenous $\beta$-tubulin but produces a small increase in the synthesis of $\alpha$-tubulin. Analysis of cells labeled to steady state showed that HA$\beta$1-tubulin accumulates to a similar level as the wild-type gene product, but together these polypeptides produce only a small increase in total tubulin content consistent with the increased synthesis of $\alpha$-tubulin. It thus appears that HA$\beta$1-tubulin successfully competes with endogenous $\beta$-tubulin for heterodimer formation and that free $\beta$-tubulin subunits (endogenous and HA$\beta$1) are selectively degraded to maintain coordinate amounts of $\alpha$- and $\beta$-tubulin. In addition, the increased synthesis of $\alpha$-tubulin suggested the existence of a mechanism to ensure coordinate synthesis of $\alpha$- and $\beta$-tubulin subunits. To analyze whether reciprocal changes in endogenous tubulin synthesis occur when $\alpha$-tubulin is overexpressed, stably transfected CHO cell lines were isolated in which HA$\alpha$1-tubulin represents 50% of the total $\alpha$-tubulin, and its relative abundance can be further increased to 85-90% by treatment with sodium butyrate. In contrast with results obtained using HA$\beta$1-tubulin, transfection of HA$\alpha$1-tubulin decreased the synthesis of endogenous $\alpha$-tubulin to 60% of normal with little or no change in $\beta$-tubulin synthesis. When the transfected cells were treated with sodium butyrate to further increase HA$\beta$1-tubulin production, a larger decrease in the synthesis of endogenous $\alpha$-tubulin (to 30% of normal) was observed. The repression on the synthesis of endogenous $\alpha$-tubulin polypeptide was found to be directly proportional to the expression of HA$\alpha$1-tubulin indicating the existence of an autoregulatory loop, where $\alpha$-tubulin inhibits its own synthesis. To determine whether overproduction of HA$\alpha$1-tubulin affected the transcription, message stability or translation of endogenous $\alpha$-tubulin, the steady state levels of $\alpha$-tubulin mRNA were analyzed by ribonuclease protection assays. The results showed that the steady state level of $\alpha$-tubulin mRNA is not affected by the overexpression of HA$\alpha$1-tubulin, indicating that the repression is translational. The results are compatible with a model in which $\beta$-tubulin synthesis is largely unperturbed by overexpression of other tubulin subunits, and excess $\beta$-tubulin subunits are rapidly degraded to maintain coordinate $\alpha$- and $\beta$-tubulin levels at steady state. In contrast, free $\alpha$-tubulin represses its own synthesis at the translational level, suggesting that its level of production may be controlled by the amount of $\beta$-tubulin available for heterodimer formation. ^

INACTIVATION OF MUTAGENIC DRUGS AND THEIR METABOLITES IN THE URINE OF PATIENTS ADMINISTERED ANTINEOPLASTIC THERAPY

Urines from patients administered mutagenic antineoplastic drugs were significantly mutagenic in the Ames assay, and hence may pose a genotoxic hazard to hospital personnel or family members caring for the patient. The urines were tested for mutagenicity in several different strains of Salmonella typhimurium that were uvr positive or negative (TA98, TA100, TA102, UTH8413, UTH8414). The urines were fractionated by high pressure liquid chromatography (HPLC) and the fractions assayed for mutagenicity in the strains in which the whole urine was mutagenic. Only fractions of urines containing the parent compound (cisplatin, doxorubicin, or mitomycin) were mutagenic; no other fraction showed significant mutagenicity. However, urine containing cyclophosphamide had two fractions that were mutagenic. One fraction, the fraction containing cyclophosphamide, required metabolic activation for mutagenicity. The other fraction did not require activation for mutagenicity.^ The chemical and mutagenic stability of these urines at room temperature was assayed over a 14 day period. The parent compound degraded within the first seven days, but the urines remained mutagenic. Cis-platinum was chemically stable in the urine; however, the urine decreased in mutagenicity. The decrease was probably the result of stable ligands binding to the platinum.^ Inactivation methods were developed to reduce the genotoxic hazard. Urine containing cisplatin was inactivated by complexing the cisplatin with diethyldithiocarbamate (DDTC). Oxidation with NaOCl of urines containing mitomycin and doxorubicin (sodium thiosulfate must be added to the doxorubicin urine) results in mutagenic inactivation. Inactivation of urine containing cyclophosphamide requires oxidation with alkaline potassium permaganate and trapping of active degradation products with sodium thiosulfate. Urines containing these drugs can be inactivated, but not always by the same method that inactivates the drug alone in solution. Therefore, in the future development of inactivation methods, both chemical and mutagenic assays are necessary to determine effectiveness. Methods of inactivation of mutagenic excreta developed in this study are both effective and practical. ^