Highly specific inhibition of IGF-I-stimulated differentiation by an antisense oligodeoxyribonucleotide to myogenin mRNA. No effects on other actions of IGF-T.

Myogenin is a member of the recently discovered family of muscle determination genes that have been shown to induce myogenic differentiation in nonmuscle cells and to be closely correlated with terminal differentiation in myoblasts. An antisense oligodeoxyribonucleotide complementary to the first five codons of myogenin blocks the stimulation of terminal myogenic differentiation by insulin-like growth factor I (IGF-I). This effect exhibits a high degree of specificity on two levels; exchanging the positions as few as 2 of the 15 bases in the oligomer abolishes its activity, and none of the other processes stimulated by IGF-I in L6A1 myoblasts are affected by the presence of the oligomer. These processes include cell proliferation as well as incorporation of leucine, uridine, and thymidine into macromolecules. The specificity, ease, and convenience of this approach indicates its potential applicability to studies on actions of other putative controlling genes in other systems.     

A rapid fluorometric method for the estimation of DNA in cultured cells

We present here a procedure for the rapid measurement of both DNA and protein from the same aliquot of cell lysate. DNA estimates obtained by this method were compared to replicate determinations using the method of Kissane and Robins (1). The optimal range for the estimation of DNA (1 to 20 μg) is well suited for use with portions of extracts from individual cell cultures; the remainder of the extract remains available for enzyme assays or other parallel determinations.     

Effects of inhibitors of ornithine and S-adenosylmethionine decarboxylases on L6 myoblast proliferation

The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C-DFMO uptake in the presence and absence of these compounds demonstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S-adenosyl-methionine-decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.     

Modulation of insulin-like growth factor actions in L6A1 myoblasts by insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5: A dual role for IGFBP-5

We have previously shown that the insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of skeletal muscle cells in culture, and that these actions in L6A1 muscle cells may be modulated by three secreted IGF binding proteins (IGFBPs), IGFBP-4, -5, and -6. Since we found that the temporal expression pattern of IGFBP-4 and IGFBP-5 differed dramatically during the transition from proliferating myoblasts to differentiated myotubes, we undertook the current study to examine the effects of purified IGFBP-4 and IGFBP-5 on IGF- stimulated actions in L6A1 muscle cells. As has been shown for other cell types, we found that IGFBP-4 had only inhibitory actions, inhibiting IGF-I and IGF-II- stimulated proliferation and differentiation. In contrast, IGFBP-5 exhibited both inhibitory and stimulatory actions. When added in the presence of 30 ng/ml IGF-I, IGFBP-5 (250 ng/ml) inhibited all markers of the early proliferative response: the tyrosine phosphorylation of the cytoplasmic signaling molecules IRS-1 and Shc, the activation of the MAP kinases, ERK1 and 2, the elevation of c-fos mRNA, the early inhibition of the elevation in myogenin mRNA, and the increase in cell number. In contrast, IGFBP-5 stimulated all aspects of the myogenic response to IGF-I: the later rise in myogenin mRNA, the elevation of creatine kinase activity, and the fusion of myoblasts into myotubes. This dual response to IGFBP-5 was greatest when it was added at a molar ratio of IGFBP-5 to IGF-I of 2:1. In contrast, when IGFBP-5 was added in the presence of IGF-II, it inhibited both proliferation and differentiation. Neither IGFBP had any effect when added in the presence of R3 IGF-I, an analog with substantially reduced affinity for IGFBPs. Our results suggest that the role of IGFBP-4 is mainly to sequester excess IGFs, and thus inhibit all actions. IGFBP-5, however, is capable of eliciting a dual response, possibly due to its unique ability to associate with the cell membrane. J. Cell. Physiol. 177:47–57, 1998. © 1998 Wiley-Liss, Inc.     

Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression.

Stimulation of myogenic differentiation by the insulin-like growth factors (IGFs) has been established for many years, but our attempts to elucidate the mechanism of that stimulation have been successful only in eliminating some likely possibilities. The recent discovery of a family of muscle determination genes has opened a new approach to this question, allowing specific focus on those genes that might play central roles in controlling myogenesis. We now report that IGF-I stimulates terminal myogenic differentiation in L6A1 cells by inducing a large increase in expression of the myogenin gene. This conclusion is supported by the following observations. 1) Myogenin mRNA is elevated by IGF-I, with a concentration dependency that parallels the stimulation of differentiation, including a decrease in stimulation at higher concentrations. 2) The time course of elevation of myogenin mRNA is consistent with its acting as an intermediate in the signalling pathway between occupancy of the IGF-I receptor and induction of expression of muscle-specific genes. 3) Inhibitors of myogenesis also inhibit elevation of myogenin mRNA in response to IGF-I. 4) An antisense oligonucleotide to the N-terminus of myogenin prevents the stimulation of differentiation by IGF-I and IGF-II, but has no effect on other actions of IGF-I on myoblasts. MyoD has been reported not to be expressed in L6 cells, and the expression of myf-5 and herculin/myf-6/MRF4 is reportedly low or undetectable. Thus, the stimulation of differentiation by IGF-I can be attributed largely, if not entirely, to increased expression of the myogenin gene. However, the relatively long time period between addition of the IGFs and elevation of myogenin mRNA as well as the inhibition of this process by several inhibitors indicate that increased myogenin mRNA levels are not a simple direct result of occupation of the IGF-I receptor.     

Polyamine depletion inhibits the differentiation of L6 myoblast cells

Exposure to alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, inhibited the insulin induced differentiation of L6 myoblast cells. Differentiation was assessed by measuring creatine kinase activity and by determining the percentage of nuclei in myotubes. The levels of putrescine and spermidine increased in stimulated cultures prior to their differentiation and these increases were blocked by alpha-difluoromethylornithine. Provision of exogenous putrescine was able to reverse the inhibitory effect of the drug. The anti-differentiative effect is observed only if alpha-difluoromethylornithine is added within twenty-four hours of insulin stimulation. In the experimental protocol used, alpha-difluoromethylornithine was added as the cultures approached confluence and had no effect on their ultimate DNA content. Therefore, the effect of alpha-difluoromethylornithine on myoblast differentiation is not secondary to an effect on cellular proliferation. These results indicate that polyamines may be involved in the mediation of muscle cell differentiation.     

A simplified method for chromatography of Dnp-leucine in the determination of intracellular specific activity of (3H) leucine

We describe a radioisotopic assay for Δ¹-pyrroline-5-carboxylate reductase. In this assay we use Δ¹-pyrroline-5-carboxylate[U-¹⁴C] and isolate product l-[U-¹⁴C]proline by cation-exchange column chromatography.     

Assay of creatine kinase in microtiter plates using thio-NAD to allow monitoring at 405 nM

An assay system for creatine kinase using microtiter plates and a plate reader that records absorbancies at 405 nM has been devised. The system is an adaptation of well-established assays that couple creatine kinase with the reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase (G6PDH), to give a measurable increase in reduced pyridine nucleotide quantitated by absorbance at 340 nM. Two features of this system are modified for reading at 405 nM: (i) The thioamido derivative of NAD is used because its reduced form exhibits a substantial increase in absorbance at 405 nM, the most commonly available wavelength on microplate readers; and (ii) glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is used because it can reduce either NAD or NADP (unlike most other G6PDH enzymes, which require NADP), thus making it unnecessary to use the more expensive thio-NADP. The rate of thio-NAD reduction is linear with enzyme concentration and time over a 20-fold range of concentrations of purified creatine kinase, and the assay also works well with myogenic cells allowed to grow and differentiate in the 96-well plate in which the assay is performed. This system offers considerable savings in cells, time, and material in studies of muscle cell differentiation, for which creatine kinase levels are frequently measured. It also provides a potential method for the convenient and economical measurement of activities of many other enzymes that can be coupled to reduction of thio-NAD.