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

Martin Kaefer, Sreenu Vemulapalli, Michael R. Freeman

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Purpose: 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. Materials and Methods: Neonatal sheep smooth muscle cell cultures were obtained from whole bladder explants. Immunohistochemical staining was performed for desmin and α-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 μg./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. Results: Primary cell cultures stained positive for desmin and α-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 μg./ml., resulting in a 40% decrease in proliferation (p <0.0001). Conclusion: 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.

Original languageEnglish
Pages (from-to)580-584
Number of pages5
JournalJournal of Urology
Volume163
Issue number2
StatePublished - Feb 2000

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Diphtheria Toxin
Epidermal Growth Factor
Smooth Muscle Myocytes
Heparin
Urinary Bladder
Cell Proliferation
Smooth Muscle
Desmin
Reverse Transcriptase Polymerase Chain Reaction
Actins
Sheep
Urinary Bladder Neck Obstruction
Primary Cell Culture
Membranes
Ataxia
Growth
Mitogens
Leucine
Hypertrophy
Cell Culture Techniques

Keywords

  • Bladder
  • Bladder neck obstruction
  • Diphtheria toxin
  • Hypertrophy
  • Muscle, smooth

ASJC Scopus subject areas

  • Urology

Cite this

A nontoxic diphtheria toxin analogue inhibits neonatal bladder smooth muscle cell proliferation. / Kaefer, Martin; Vemulapalli, Sreenu; Freeman, Michael R.

In: Journal of Urology, Vol. 163, No. 2, 02.2000, p. 580-584.

Research output: Contribution to journalArticle

Kaefer, Martin ; Vemulapalli, Sreenu ; Freeman, Michael R. / A nontoxic diphtheria toxin analogue inhibits neonatal bladder smooth muscle cell proliferation. In: Journal of Urology. 2000 ; Vol. 163, No. 2. pp. 580-584.
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abstract = "Purpose: 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. Materials and Methods: Neonatal sheep smooth muscle cell cultures were obtained from whole bladder explants. Immunohistochemical staining was performed for desmin and α-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 μg./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. Results: Primary cell cultures stained positive for desmin and α-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 μg./ml., resulting in a 40{\%} decrease in proliferation (p <0.0001). Conclusion: 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.",
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N2 - Purpose: 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. Materials and Methods: Neonatal sheep smooth muscle cell cultures were obtained from whole bladder explants. Immunohistochemical staining was performed for desmin and α-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 μg./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. Results: Primary cell cultures stained positive for desmin and α-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 μg./ml., resulting in a 40% decrease in proliferation (p <0.0001). Conclusion: 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.

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