A nontoxic diphtheria toxin analogue inhibits neonatal bladder smooth muscle cell proliferation

The response of the neonatal bladder to infravesical obstruction, such as posterior urethral valves or detrusor-sphincter dyssynergia, may result in structural and functional changes that persist well after obstruction is treated. A pharmacological means of inhibiting smooth muscle cell proliferation would likely serve to halt or reverse this deleterious process. Heparin binding (HB) epidermal growth factor (EGF) is a known smooth muscle cell mitogen, while its membrane bound precursor serves as the diphtheria toxin receptor. We report the effects of the nontoxic diphtheria toxin analogue cross reacting material (CRM) 197 on neonatal sheep bladder smooth muscle cell proliferation.

Neonatal sheep smooth muscle cell cultures were obtained from whole bladder explants. Immunohistochemical staining was performed for desmin and alpha-smooth muscle actin. HB-EGF messenger RNA was detected by reverse transcriptase polymerase chain reaction using primers to the human sequence, while pro-HB-EGF peptide was confirmed using a diphtheria toxin sensitivity assay with incorporation of tritiated leucine. Cells grown in 96 well plates were exposed to 1, 10 and 100 microg./ml. CRM 197 for 5 days, after which relative cell number was determined using an 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide; thiazolyl blue based assay read at a wavelength of 550 nm. Statistical analysis was performed using Student's t test.

Primary cell cultures stained positive for desmin and alpha-smooth muscle actin, confirming a smooth muscle origin. Reverse transcriptase polymerase chain reaction yielded a 453 bp product with 88% homology to human HB-EGF. Total protein synthesis significantly decreased when cells were incubated with diphtheria toxin, confirming the presence of membrane bound pro-HB-EGF. CRM 197 inhibited bladder smooth muscle cell growth in a dose dependent fashion at a concentration of 10 microg./ml., resulting in a 40% decrease in proliferation (p <0.0001).

CRM 197 inhibits bladder smooth muscle cell proliferation in a dose dependent, nontoxic fashion through its interaction with HB-EGF. These data suggest that molecular strategies designed to inhibit HB-EGF mediated cell growth may prove beneficial for the prevention and/or treatment of detrusor hypertrophy secondary to anatomical or functional bladder outlet obstruction.