Biomedical subjects
Publications and source records attributed to A K Verma.
Explore the source record for details and available documents.
Mouse skin has a long history as a useful model for the study of the mechanism of carcinogenesis (6). In particular, the availability of specific diterpene esters has made possible rapid progress in understanding the mechanism of tumor formation (4,6,8,19,36,41), although certain details may be unique to promotion by phorbol esters. Evidence is compatible with an essential role for elevated levels of polyamines in tumor promotion, but other components of phorbol ester action on mouse skin are also essential (27,40,54). These may include the production of dark cells (22), inhibition of maturation (2,19,41), and the elimination of metabolic cooperation (12,57). Factors modifying biochemical processes that are essential to tumor formation produce a parallel effect on tumor formation. Some of these inhibitors act synergistically to inhibit tumor formation (50,55), and knowledge of their action may lead to practical application for the prevention of human cancer.
The biology of tumor formation by the initiation-promotion protocol differs from that of the complete carcinogenesis process. In the latter case, the latency period is longer and tumor yield is less, but carcinomas appear much earlier. Retinoic acid, a potent inhibitor of both the induction of ODC activity and tumor promotion by TPA, failed to inhibit both the induction of ODC activity and tumor formation by DMBA. 7,8-Benzoflavone, which did not inhibit the induction of ODC activity by TPA, inhibited the induction of ODC activity and tumor formation by DMBA. The results indicate that: (a) mechanism of the induction of ODC activity and tumor formation by a complete carcinogen appears to be different from that of the tumor promoter TPA; (b) DMBA-induced ODC activity may be an important component of the mechanism of DMBA carcinogenesis; and (c) although there is a wealth of data that indicate the efficacy of the retinoids in the prevention of a variety of cancers in experimental animals, including mammary carcinogenesis by DMBA (3,5), the present results and those reported by others (2) are not in agreement with a universal effect of retinoic acid in the prevention of carcinogenesis.
Arginase, which catalyzes the cleavage of L-arginine to urea and ornithine, was detected in both soluble and particulate fractions of mouse epidermis. In a typical experiment, about 75 and 25% of the total arginase activity was associated with the soluble (100 000 X g supernatant) and the washed particulate fraction, respectively. Both soluble and particulate enzymes required the presence of divalent Mn2+ for activity. Arginase activity was increased by about 50% in the particulate fraction, but not in the soluble fraction, by preheating the fractions at either 50 or 55 degrees C in the presence of 15 mM MnCl2. Enzyme activity in both fractions, in the absence of 15 mM MnCl2, dropped precipitously during heating. A comparison of the nature of arginases in the soluble and particulate fractions revealed similar Km values (13 mM) and pH optima (9.5) and identical heat denaturation curves. Application of 10 nmol of 12-O-tetradecanoylphorbol-13-acetate to mouse skin did not increase arginase activity in either fraction over a period of 24 h. In contrast, there was a large increase in ornithine decarboxylase activity in the soluble fraction 4.5 h after treatment. Mouse epidermal ornithine decarboxylase activity was much less than arginase activity and was predominantly localized in the soluble fraction. These results indicate that the normal level of arginase activity is not a limiting factor for the stimulation of polyamine biosynthesis by TPA. High arginase activity in mouse epidermis may play a role in providing ornithine for polyamine biosynthesis and in the production of glutamate and proline as well as in the production of keratinous proteins.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Plasma membrane fractions from rat corpus luteum contain two kinds of Ca2+-stimulated ATPase, one having a high affinity for Ca2+, the other a low affinity for Ca2+. The high affinity ATPase had a specific Ca2+ requirement with a K 1/2 of 0.2 to 0.3 microM; it had a Vmax of 105 nmol min-1 mg-1 and distributed, upon subcellular fractionation, with recognized plasma membrane enzymes. The properties of this enzyme indicate that it is a CA2+ extrusion pump. The low affinity pump (K 1/2 for Ca2+, about 15 microM) was nonspecific, being stimulated equally well by Ca2+ of Mg2+; its function is unknown. Although the high affinity ATPase resembled the erythrocyte Ca2+-pumping ATPase in the properties mentioned above, it differed in that it failed to respond to Mg2+ or calmodulin. The lack of response to Mg2+ was due to the enzyme's retention of endogenous Mg2+; it did, after incubation with chelators, show a Mg2+ requirement. However, we were unable to show any effect of added calmodulin or trifluoperazine. This failure may be related to the high content of tightly bound calmodulin in these membranes. Much of this calmodulin could not be extracted even by washing with 1 mM EGTA and/or 0.1% (w/v) Triton X-100. This enzyme, the erythrocyte enzyme, and the adipocyte plasma membrane Ca2+ ATPase all belong to the class of Ca2+ ATPases with plasma membrane distribution and high affinity for Ca2+, indicating that they are Ca2+ extrusion pumps. However, the data indicate that tissue-specific differences exist within this class, with the enzyme from adipocytes and rat corpus luteum belonging to a subclass in which the requirement for Mg2+ and any response to calmodulin are difficult to demonstrate.
Explore the source record for details and available documents.
Previous studies have established the presence of polypeptide hormone receptors in Golgi fractions from rodent liver. In this study we attempted to identify peptide hormone receptors in other intracellular elements, particularly lysosomes. Tritosomes were prepared by a standard procedure, and highly purified secondary lysosomes were prepared by fractionating the L fraction of rat liver in a discontinuous metrizamide gradient into subfractions L1 to L4. Binding of 125I-labeled insulin and 125I-labeled somatotropin was studied with membranes prepared from osmotically shocked fractions. The L2 and L3 fractions, virtually devoid of galactosyltransferase (UDP galactose:2-acetamido-2-deoxy-D-glucosylglycopeptide galactosyltransferase, EC 2.4.1.38) but highly enriched in acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2], appeared as classical secondary lysosomes by electron microscopy. When compared with Golgi fractions, the level of specific binding per 50 micrograms of protein of 125I-labeled somatotropin in L2 and L3 was 1/3, whereas that of 125I-labeled insulin was comparable. L1, which was reduced in acid phosphatase and increased in galactosyltransferase activities, showed higher hormone binding than did L2 and L3. This was not attributable to Golgi fraction contamination, as evident by specific binding/galactosyltransferase ratios. Binding to tritosome membranes could be largely accounted for by variable contamination with Golgi fractions as judged by specific binding/galactosyltransferase ratios. To clarify the distribution of receptor sites in lysosomal preparations, we fractionated the entire L fraction on a continuous Percoll gradient. Acid phosphatase and galactosyltransferase activities were segregated to the high and low density ranges of the gradient, respectively; however, the fractions enriched in hormone binding were of intermediate density, distinct from Golgi and lysosomal biochemical markers. We conclude that intracellular receptors are found not only in galactosyltransferase-containing very low density lipoprotein-marked Golgi vesicles but also in a unique vesicle of intermediate density between classical Golgi and lysosomal structures.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Application of a single large dose (3.6 micromol) or smaller weekly repeated doses (0.2 micromol) of 7,12-dimethylbenz[a]anthracene (DMBA) to the skin of CD-1 mice led to a 20 to 50-fold increase in epidermal ornithine decarboxylase (ODC) (EC 4.1.1.17) activity as well as tumor formation. Retinoic acid (0.17-68 nmol), a potent inhibitor of both the induction of ODC activity and tumor formation by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), failed to inhibit both the induction of ODC activity and tumor formation by DMBA. In contrast, 7,8-benzoflavone (367 nmol), which did not inhibit the induction of ODC activity by TPA, effectively inhibited the induction of ODC activity as well as the formation of skin tumors caused by DMBA. These results indicate that (a) the mechanism of the induction of ODC activity and tumor formation by a complete carcinogen appears to be different from that of the tumor promoter TPA, (b) DMBA-induced ODC activity may be an important component of the mechanism of DMBA carcinogenesis, and (c) the protective effect of retinoic acid on skin carcinogenesis is not universal; it inhibits skin tumor formation by some agents and not by others.
Explore the source record for details and available documents.
The ability of 5,6-epoxyretinoic acid, a biologically active metabolites of retinoic acid, to inhibit both the induction of ornithine decarboxylase (ODC) activity and skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) was evaluated. Application of 5,6-epoxyretinoic acid either concurrently with or 1 hr after each application of TPA to the initiated mouse skin inhibited the formation of skin tumors as effectively as did retinoic acid. 5,6-Dihydroretinoic acid, which is a poor substrate for epoxidation, also inhibited skin tumor promotion. 5,6-Epoxyretinoic acid, 5,6-dihydroretinoic acid, and retinoic acid were equally effective in inhibiting the induction of ODC activity by TPA. Insect juvenile hormones inhibited neither the induction of ODC activity nor skin tumor promotion by TPA. These results indicate that (a) epoxidation of retinoic acid at the 5,6-position is not a rate-limiting modification for the anti-promoting activity of retinoic acid and that (b) inhibition of the induction by TPA of mouse epidermal ODC activity may be a simple test for screening the potential prophylactic activities of new retinoids.
The induction of epidermal ornithine decarboxylase (EC 4.1.1.17) (ODC) following topical application of the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) to mice can be inhibited by topical application of putrescine, the product of the enzyme. The degree of inhibition depended on both the dose and the time of putrescine application; application of 20 mumol of putrescine 2 hr after TPA treatment inhibited the induction of ODC activity by 50%. TPA-induced activity of another polyamine-biosynthetic enzyme, S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50), was unaffected by application of putrescine. Among several amines tested for their ability to inhibit the induction of ODC activity, spermidine, 1,7-diaminoheptane, and spermine were the most effective, causing a 90% inhibition at the 20-mumol dose. Putrescine, when added directly to the assay medium at a 100-mumol dose level inhibited by 97% the TPA-induced ODC activity, but the amount of putrescine (20 mumol) which gave 50% inhibition of the induction of ODC activity in vivo had no effect when added to the assay system. Mixing of soluble extracts from TPA-treated mouse epidermis posttreated either with acetone or putrescine or with mouse epidermis treated with putrescine alone gave essentially additive ODC activity. Furthermore, putrescine did not elicit production of detectable ODC-antizyme activity in mouse epidermis. Putrescine inhibited the formation of mouse skin papillomas promoted with TPA. Topical application of 20 and 100 mumol of putrescine 2 hr after each application of TPA to mice initiated with 7,12-dimethylbenz[a]anthracene resulted in a 30 and 80% inhibition, respectively, of papilloma formation compared to animals receiving no putrescine.
Explore the source record for details and available documents.
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the antileukemic agent mezerein are diterpene esters of plant origin with certain structural similarities. Both compounds, when applied topically to mouse skin, were equipotent on a molar basis in inducing hyperplasia, inflammation, and ornithine decarboxylase activity, as well as in reducing cyclic adenosine 3':5'-monophosphate accumulation in response to beta-adrenergic stimulation. In contrast, mezerein was much less effective as a tumor promoter; the phorbol ester at 8.5 nmol/application yielded 78-fold more tumors than did 8.5 nmol mezerein per application to similarly initiated SENCAR mice. The superiority of the phorbol ester was nearly as great in CD-1 mice.
Explore the source record for details and available documents.