Safety and efficacy of deoxycytidine/deoxythymidine combination therapy in POLG-related disorders: 6-month interim results of an open-label, single arm, phase 2 trial.

Background: DNA polymerase gamma (POLG)-related disorders are a group of rare neurodegenerative mitochondrial diseases caused by pathogenic variants in POLG, the gene encoding POLG. Patients may experience a range of signs and symptoms, including seizures, vision loss, myopathy, neuropathy, developmental impairment or regression, and liver failure. The diseases follow a progressive, degenerative course, with most affected individuals dying within 3 months-12 years of diagnosis. At present, there are no effective treatments for POLG-related disorders. Methods: In this study we report the interim 6-month data from a long term open-label, single arm phase 2 trial, in which we assessed the safety and efficacy of combination therapy with deoxycytidine and deoxythymidine (dC/dT) in children with POLG-related disorders. dC/dT was given enterally in powder form, dissolved in water. The primary outcome measures included Newcastle Mitochondrial Disease Scale (NMDS) score, serum growth differentiation factor 15 (GDF-15; a biomarker of mitochondrial dysfunction), electroencephalography (EEG), seizure diary, and blood and urine tests to assess end organ and mitochondrial function. Secondary outcome measures included recording of all adverse events to evaluate the safety of the intervention. The trial is registered with ClinicalTrials.gov, NCT04802707 (https://clinicaltrials.gov/ct2/show/NCT04802707). Data were collected from 14 October, 2021 to 13 December, 2023. Findings: We present 6-month interim data from the first ten people with POLG-related disorders enrolled in the trial, six with Alpers-Huttenlocher syndrome, two with ataxia-neuropathy spectrum, and two who do not fit into a classical POLG-related phenotype. During the 6 months of treatment, NMDS score improved from a mean of 27.3 at baseline to 20.7 at 6 months (estimated difference 6.0; 95% CI 2.5-∞). GDF-15 values remained stable or decreased in all patients; the mean decreased from 1031 pg/ml to 729 pg/ml (estimated difference 200; 95% CI 12-∞). 8/10 patients had abnormal baseline EEG; improvement in EEG was seen in 5 of these 8. There were no significant changes in other blood and urine testing. Regarding adverse events, two patients experienced diarrhea that spontaneously resolved. Interpretation: dC/dT is a promising treatment option for people with POLG-related disorders. Further research is needed to assess the long-term safety and efficacy in POLG-related disorders, as well as safety and efficacy in other mitochondrial DNA depletion disorders. Funding: This study was primarily funded by the Liam Foundation, with additional funding from the Savoy Foundation, Grand Défi Pierre Lavoie Foundation, and Fonds de Recherche du Québec - Santé.

CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation.

Using inverse polymerase chain reaction, we identified CD44, located on chromosome 11p13, as a novel translocation partner of IGH in 9 of 114 cases of gastric, nongastric extranodal, follicular, and nodal diffuse large B-cell lymphoma (DLBCL). Notably, these translocations involving IGHSmu were detected in follicular lymphomas and exclusively in germinal center B cell-ike (GCB)-DLBCLs. CD44 is not expressed in reactive GC B cells. The IGHSmu/CD44 translocations substitute Smu for the CD44 promoter and remove exon 1 of CD44, resulting in the overexpression of Imu-CD44 hybrid mRNA transcripts activated from derivative 11 that encode a new CD44 variant lacking the leader peptide and with a unique C-terminus (CD44DeltaEx1). When overexpressed in vitro in the CD44(-) GCB-DLBCL cell line BJAB, CD44DeltaEx1-green fluorescent protein localized to the cytoplasm and nucleus, whereas CD44s-green fluorescent protein (standard form) localized to the plasma membrane. The ectopic expression of CD44DeltaEx1 in BJAB cells enhanced their proliferation rate and clonogenic ability, indicating a possible pathogenic role of the translocation.

Antisense c-myc retroviral vector suppresses established human prostate cancer.

Prostate cancer eventually becomes androgen resistant, resumes growth, and kills the patient. Characterization of genetic events that lead to androgen refractory prostatic neoplasia has revealed the frequent overexpression of c-myc and uncontrolled prostate cancer proliferation. A novel strategy to combat advanced prostate cancer utilized a replication incompetent retrovirus that contained the mouse mammary tumor virus (MMTV) promoter within the retroviral vector to allow transcription of antisense c-myc gene within target prostate tumor cells. The transduction of cultured DU145 cells by XM6:MMTV-antisense c-myc RNA retrovirus did not affect cell proliferation in culture, yet a single direct injection of MMTV-antisense c-myc viral media into established DU145 tumors in nude mice produced a 94.5% reduction in tumor size compared to tumors treated with control virus MTMV sense fos and untreated tumor by 70 days. Two animals in the antisense c-myc-treated group had complete regression of their tumors. Histopathological examination of the tumors revealed that MMTV-antisense c-myc-transduced DU145 tumors had increased tumor cell differentiation, decreased invasion, and a marked stromal response. The mechanism for the antitumor effect of MMTV-antisense c-myc retrovirus appears to be suppression of c-myc mRNA and protein, and decreased bcl-2 protein. The in vivo transduction of prostate cancer cells with MMTV-antisense c-myc retroviruses reduced tumor growth by suppressing c-myc, resulting in the down-regulation of bcl-2 protein. Consequently, the MMTV-antisense c-myc retrovirus may be useful for gene therapy against advanced, hormone-refractory prostate cancer.

Actions of the testicular paracrine factor (P-Mod-S) on Sertoli cell transferrin secretion throughout pubertal development.

Peritubular cells that surround the seminiferous tubules have been shown to produce a paracrine factor, termed P-Mod-S, that has dramatic effects on Sertoli cell function in vitro and is postulated to be important in the control of testicular function. The current study was designed to determine whether P-Mod-S has the ability to regulate Sertoli cell function during pubertal development. Sertoli cells were isolated from 10-, 20-, and 35-day-old rats which correspond to the prepubertal, mid-pubertal, and late pubertal stages of development. Histochemical analysis of cultured cells isolated from each age group was performed to establish the purity of the cell populations used. Testicular transferrin production by Sertoli cells was used as a marker of cellular differentiation. Basal production of transferrin by the cultured cells was found to increase during the pubertal period. P-Mod-S stimulated transferrin production by Sertoli cells isolated from 10-, 20-, and 35-day-old rats. FSH appears to enhance the ability of Sertoli cells to respond to P-Mod-S with cells obtained from 10-day-old rats. Sertoli cells from 35-day-old rats were nonresponsive to regulatory agents such as FSH. P-Mod-S alone, however, significantly stimulated transferrin production by Sertoli cells from this more adult stage of development. P-Mod-S was the only individual regulatory agent tested that could stimulate transferrin production by Sertoli cells from 35-day-old rats. Results indicate that P-Mod-S has the ability to regulate Sertoli cell function throughout pubertal development. Observations suggest that P-Mod-S and FSH may act together in the prepubertal testis to promote Sertoli cell differentiation and that P-Mod-S may act in the adult testis to maintain optimal Sertoli cell function and differentiation.

Investigations on the relationship between sperm fertilizing ability and androgen-binding protein in the restricted rat.

The hypothesized relationship between androgen-binding protein (ABP) and sperm maturation was investigated using a mutant rodent: the restricted rat. The seminiferous epithelium of these animals undergoes a spontaneous degeneration, but changes are progressive. Restricted rats in the transition to infertility were used to determine if changes in ABP were related to the decreased fertility found in these animals. Fertilizing ability was determined by insemination of cauda epididymal spermatozoa into hormonally primed female rats and examination of ova for evidence of fertilization 48 h later. Epididymal and testicular tissues were analyzed for ABP using a charcoal assay. Androgen levels were determined by RIA. Testicular weights were significantly reduced compared to those of normal littermates in restricted rats at all ages; epididymal weights were significantly reduced in rats 140 days and older. Among restricted rats, sperm fertilizing ability was variable, but was significantly lower than that in normal littermates; it was consistently highest at 90 days of age. Epididymal ABP content (picomoles per organ) was significantly reduced in restricted rats at all ages; peak values occurred at 90 days. Testicular ABP content was significantly reduced only in the youngest and oldest animals. Plasma testosterone levels were not statistically lower than those found in normal littermates, and ventral prostate weights were maintained at normal levels in all four groups of animals. A significant positive correlation existed between sperm fertilizing ability and epididymal ABP, but not between sperm fertilizing ability and plasma testosterone. Since ABP is an index of Sertoli cell function, these data indicate that sperm fertilizing ability is closely related to Sertoli cell function and/or ABP.

Investigations on the relationship between sperm fertilizing ability and androgen-binding protein in the hypophysectomized, pregnenoloneenolone-injected rat.

Hypophysectomized rats maintained with 2 mg pregnenolone have low plasma testosterone levels, but rete testis levels are normal. This model system was used to examine the importance of androgen-binding protein (ABP) in maintaining high luminal androgen concentrations for the development and maintenance of sperm fertilizing ability in the epididymis. Hypophysectomized rats were injected with pregnenolone (1, 0.5, 0.2, or 0.02 mg/100 g BW) for 14 days, starting 1 day after surgery. Sham-operated rats and a group of hypophysectomized rats were injected with oils as controls. At the end of the experimental period, sperm fertilizing ability, tissue weights, ABP, and testosterone levels were determined. The 1- and 0.5-mg doses of pregnenolone resulted in rete testis testosterone levels that were 67% and 29%, respectively, of the levels found in sham-operated animals. Plasma testosterone levels were not different from levels found in hypophysectomized, oil-injected controls with any of the pregnenolone doses. The three highest doses of pregnenolone resulted in levels of testicular ABP that were not statistically different from sham-operated levels (2 pmol/organ). Epididymal ABP content was maintained at sham-operated control levels (30 pmol/organ) with the 1-mg dose; with the 0.5- and 0.2-mg doses, ABP levels were 76% and 73% of sham-operated levels, respectively. Epididymal ABP specific content (picomoles per 100 mg tissue) was maintained at sham-operated control levels with the 1-, 0.5-, and 0.2-mg doses. Sperm fertilizing ability was maintained at sham-operated control levels with the three highest doses of pregnenolone. The 0.02-mg dose of pregnenolone resulted in values that were not statistically different from hypophysectomized, oil-injected control values for all parameters tested. A significant positive correlation existed between ABP and sperm fertilizing ability. These data demonstrate that testicular and epididymal ABP levels can be maintained in hypophysectomized rats with pregnenolone treatment alone, and that sperm fertilizing ability can be maintained when intraluminal androgen levels are low and ABP levels are near normal. This suggests that ABP may be important in the maintenance of normal sperm fertilizing ability.

Regulation of Sertoli cell differentiated function: testicular transferrin and androgen-binding protein expression.

The regulation of Sertoli cell function was investigated through an examination of the effects of various hormones, regulatory agents, and culture conditions on testicular transferrin and androgen-binding protein (ABP) synthesis and steady state levels of mRNA. FSH stimulated both transferrin and ABP production 2-fold above control levels. Interestingly, FSH had a differential effect on transferrin and ABP mRNA levels, with 1.25- and 2.0-fold respective increases in steady state levels of mRNA. Insulin and retinol stimulated both transferrin and ABP synthesis in a similar manner. Testosterone had no significant effect on either transferrin or ABP mRNA levels or synthesis. Maximum stimulation of both transferrin and ABP production occurred when Sertoli cell cultures were treated with a combination of FSH, insulin, and retinol, which resulted in a greater than 4-fold stimulation of synthesis and 2-fold stimulation of gene expression. Optimal transferrin and ABP secretion occurred between days 4-6 of Sertoli cell culture and subsequently declined. Sertoli cell number decreased with time in culture, such that approximately a 50% loss of cells was observed after 10 days of culture. The responsiveness of Sertoli cells to regulatory agents was altered by cell density, with a maximum responsiveness achieved at a density of 12 micrograms DNA/2 cm2 for both transferrin and ABP. As the cell density deviated from this level the responsiveness of cells to regulatory agents decreased and approached control values. These observations indicate that the culture conditions and the method of data normalization are important parameters in an analysis of the hormonal regulation of Sertoli cell function. FSH actions on Sertoli cells increased both cellular and excreted cAMP levels but had no effect on cGMP levels. (Bu)2 cAMP affected transferrin and ABP mRNA levels and synthesis in a similar manner, with approximately a 3-fold increase in synthesis and a 1.5-fold increase in steady state levels of mRNA. The minimum and maximum effective concentrations of (Bu)2AMP for both proteins were 1 and 10 microM, respectively. Observations imply that regulatory agents that act via a cAMP-mediated signal transduction mechanism, such as FSH, will probably have similar actions on transferrin and ABP production. In addition, data obtained with insulin and retinol indicate that transferrin and ABP production can be similarly regulated with cAMP-independent signal transduction mechanisms. Results indicate that transferrin and ABP mRNA levels and synthesis are regulated in a coordinate manner with the regulatory agents and culture conditions evaluated.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of the androgen receptor in isolated testicular cell types with a microassay that uses an affinity ligand.

A microassay for the androgen receptor was developed to investigate the cellular distribution of receptor in freshly isolated testicular cell types. The microassay uses an androgen affinity ligand, 17 beta-dihydrotestosterone bromoacetate. Binding of this ligand by the androgen receptor is rapid and irreversible, which permits the development of a highly sensitive assay. The androgen receptor microassay is completed within 4 h and detects receptor in as little as 0.5 micrograms cellular protein. There was no detectable binding of the affinity label by albumin or Sertoli cell-secreted proteins, including androgen-binding protein. Androgen receptor was found in cellular sonicates of human foreskin fibroblast, rat ventral prostate, rat kidney, and rat liver. Although the relative distribution of receptor was similar to that obtained using a traditional equilibrium binding assay, the levels of receptor were significantly higher using the microassay. The androgen receptor microassay was subsequently used to investigate the receptor in isolated testicular cell types. Androgen receptor was detected in freshly isolated peritubular myoid cells (80 fmol/micrograms DNA), Sertoli cells (88 fmol/micrograms DNA), and Leydig cells (35 fmol/micrograms DNA). No androgen receptor was detected in a mixed population of germ cells. Hormones were not found to influence androgen receptor levels in cultured peritubular cells or Sertoli cells. Electrophoretic analysis of androgen receptor radiolabeled with the affinity ligand demonstrates a single 52-kDa form of the receptor in peritubular cells, Sertoli cells, and Leydig cells. The size of the androgen receptor species detected in the rat testicular cell types was slightly smaller than the 56-kDa protein detected in a human fibroblast cell line. The current study demonstrates the utility of the microassay and affinity ligand to investigate androgen receptor biology. Data indicate that androgen receptors are present in several testicular cell types and suggest that the control of testicular function by androgens probably involves actions on multiple cell types.

Progressive nuclear translocation of somatostatin analogs.

UNLABELLED: Optimal cancer radiotherapy using Auger electron emitters requires selective localization of radionuclides in close proximity to tumor DNA. METHODS: Intracellular trafficking of (125)I-Tyr1-somatostatin-14 somatotropin-release inhibiting factor (SRIF) and 2 of its analogs, (125)I-WOC 4a and (111)In-pentetreotide, was studied in human neuroblastoma cells. RESULTS: After 24-h incubation, SRIF was degraded or recycled, whereas its protease-resistant analogs progressively accumulated in nuclear fractions. (111)In-pentetreotide binding to DNA increased over time in somatostatin receptor-positive cells but not in somatostatin receptor-negative cells. CONCLUSION: These in vitro studies show that prolonged exposure to radiolabeled SRIF analogs significantly increases their cellular internalization, nuclear translocation, and DNA binding. Clinically, infusion of radiolabeled somatostatin analogs may enhance tumor uptake and retention and provide more effective in situ radiotherapy.

Cellular localization of fibronectin gene expression in the seminiferous tubule.

The cellular location of fibronectin expression within the seminiferous tubule was investigated in order to better understand testicular cell functions and cell-cell interactions. Peritubular cells were shown to actively synthesize and secrete fibronectin in culture by the detection of a radiolabeled 220 kDa secreted protein that is immunologically similar to fibronectin and by the quantitation of fibronectin in peritubular cell conditioned medium with a fibronectin enzyme-linked immunosorbent assay. Sertoli cells did not produce detectable levels of fibronectin when assayed by either of these procedures. A 6.5 kb fibronectin messenger RNA was detected in freshly isolated or cultured peritubular cells, but no fibronectin gene expression was detected in Sertoli cells or developing germinal cells. Combined results imply that the peritubular cells are the only apparent site of fibronectin expression within the seminiferous tubule. During the development of the testis the levels of fibronectin expression increased to a maximum at early puberty (15-day-old rats) and then slowly declined. The results demonstrate that fibronectin can be utilized as a unique functional and biochemical marker for peritubular cells when compared to other cell types in the seminiferous tubule. Production of fibronectin by peritubular cells provides an example of the ability of peritubular cells and Sertoli cells to cooperate in the production of individual components of the basement membrane of the seminiferous tubule. This cellular interaction is an example of a mesenchymal/stromal-epithelial interaction which is postulated to be important for the physiology of many tissues.

Cell-cell interactions and the regulation of testis function.

Regulatory interactions have been shown to occur between all the testicular cell types considered. The paracrine factors mediating these interactions generally influence either cellular growth or differentiation. The regulation of cellular growth is essential in the developing testis and is required for the maintenance of spermatogenesis in the adult testis. The rapid rate of germinal cell proliferation and the continuous but slowed growth of the peritubular cells and Leydig cells requires the presence of specific growth factors in the adult. Therefore, cell-cell interactions have evolved that involve growth factors such as IGF, TGF-alpha, TGF-beta and NGF. Other growth factors such as FGF or less characterized components like the seminiferous growth factor (SGF) also may be involved in the paracrine regulation of testis cell growth. An alternate cellular parameter to cell growth to consider is the regulation of cellular function and differentiation. A number of endocrine agents and locally produced paracrine factors have been shown to control and maintain testis cell function and differentiation. Cell-cell interactions mediated by factors such as androgens, POMC peptides, and PModS are all primarily directed at the regulation of cellular differentiation. Therefore, the agents which mediate cell-cell interactions in the testis can generally be categorized into factors that regulate cell growth or those which influence cellular differentiation. The specific cell-cell interactions identified will likely be the first of a large number of cellular interactions yet to be investigated. Although a number of potentially important cell-cell interactions have been identified, future research will require the elucidation of the in vivo physiological significance of these interactions. The existence of different cell types and potential cell-cell interactions in a tissue implies that the actions of an endocrine agent on a tissue will not simply involve a single hormone and single cell. The endocrine regulation of testis function will have effects on cell-cell interactions and be affected by local cell-cell interactions. The ability of LH to influence Leydig cell androgen production promotes a cascade of interactions mediated through several cell types to maintain the process of spermatogenesis. FSH actions on Sertoli cells also promote cell-cell interactions that influence germinal cell development, peritubular myoid cell differentiation and Leydig cell function. Therefore, elucidation of the endocrine regulation of testis function requires an understanding of the local cell-cell interactions in the testis.

Transforming growth factor beta as a clinical biomarker for prostate cancer.

OBJECTIVES: Tumor biomarkers to detect prostate cancer earlier may reduce prostate cancer deaths. Transforming growth factor-beta1 and -beta2 (TGF-beta1 and -beta2) become overexpressed in prostate cancer and might be useful tumor markers of prostate cancer. METHODS: Plasma and urinary TGF-beta1 and plasma TGF-beta2 levels were studied preoperatively in 74 consecutive patients who had prostate cancer and underwent radical prostatectomy and were compared with those of 29 similarly aged male control patients who had no clinical evidence of prostate cancer. RESULTS: Plasma TGF-beta1 levels were similar in both prostate cancer and control groups and did not correlate with serum prostate-specific antigen (PSA), clinical and pathologic stages, or Gleason grade. Urinary TGF-beta1 levels, however, increased 3.5-fold in patients with prostate cancer relative to controls and tended to be higher with advancing clinical and pathologic stages. Plasma TGF-beta2 levels, like plasma TGF-beta1 levels, were similar for both the study and control groups, but when stratified by pathologic stage or Gleason grade, patients with prostate cancer with pathologic Stage T2a and Gleason grade of 3 or less had significantly increased plasma TGF-beta2 levels as compared with either control patients or patients with prostate cancer with pathologic Stages T2b/T2c and T3/T4 or Gleason grade of 4 or more, suggesting that early prostate cancer may contribute to plasma TGF-beta2 levels. CONCLUSIONS: Unlike plasma TGF-beta1 levels, urinary TGF-beta1 and plasma TGF-beta2 levels were higher in patients with prostate cancer and may be useful biomarkers of prostate cancer.

Prostate cancer cells lose their sensitivity to TGFßI growth inhibition with tumor progression.

The observation that advanced prostate cancer has reduced sensitivity to transforming growth factor ßI (TGFßI) growth inhibition suggests that the acquisition of TGFßI resistance may play a role in prostate tumor progression. Using the Dunning R3327 rat prostate adenocarcinoma model, it was determined that prostate carcinoma cells became less responsive to TGFßI growth inhibition with differentiated tumors more resistant to TGFßI. A TGFß receptor defect was not found in advanced prostate carcinoma cells because both the type I and II TGFß receptors were present and functional. Moreover, TGFα/epidermal growth factor receptor (EGFR) and basic fibroblast growth factor (bFGF)/fibroblast growth factor receptor (FGFR) autocrine stimulatory pathways, which may potentially counter-regulate the inhibitory effects of TGFßI, were not present with prostate cancer progression. However, the likelihood that other prereceptor stimulatory pathways or TGFß postreceptor signaling alterations are responsible for reduced sensitivity to TGFßI growth inhibition remains to be elucidated.

Antiangiogenic therapy with somatostatin receptor-mediated in situ radiation.

Tumor growth and the development of metastases require an angiogenic response. Angiogenic vessels uniquely express somatostatin subtype 2 (sst 2) receptors that can transport somatostatin or its analogs into the cell. We hypothesized that radiolabeled somatostatin analogs could inhibit the angiogenic response by selectively destroying proliferating endothelial cells. We evaluated the antiangiogenic effects of 111In-pentetreotide, an sst 2-preferring somatostatin analog in a human vessel model. Disks of human placental vein were embedded in fibrin gels in culture and observed for angiogenic sprouting for 14 days. Vein disks were treated with 111In-pentetreotide (1.5, 15, and 150 microCi/mL) on the day of implantation. Control groups included disks treated with nutrient medium alone, with 111In-chloride, and with unlabeled pentetreotide. The percentage of wells that initiated an angiogenic response and the overall length and density of neovessel sprouts were assessed on Day 14. 111In-pentetreotide treatment did not completely block initiation of the angiogenic response but significantly decreased the growth of neovessels after initiation. Both the receptor-specific Auger electron-induced and nonspecific gamma radiation-mediated effects contributed to the angiotoxicity.

Compassion and patient centred care.

BACKGROUND: The essence of medicine is a relationship based upon a concern for suffering. Western medicine, arising from a modernistic philosophy, has a tradition of paternalistic 'doctor centred' care. There are significant criticisms of this approach. OBJECTIVE: Drawing on postmodern perspectives, this article discusses the nature of patient centred care. Patient centred care requires a reconciliation of the patient's and doctor's agenda via attention to communication, power and patient autonomy. Patient centred care has been defined by six domains: the illness experience, the context, finding common ground, partnership, health promotion, and consultation limitations. DISCUSSION: Patients strongly desire patient centred care. It has been associated with improved patient and doctor satisfaction, greater compliance, fewer investigations, referrals and malpractice complaints, and no change in consultation time. Patient centred care exerts a positive influence on health outcomes and is especially applicable in general practice, providing an efficacious and compassionate response to suffering.

Colchicine and 2-methoxyestradiol Inhibit Human Angiogenesis.

BACKGROUND: Angiogenesis is a critical determinant of tumor growth and the development of metastases. Tubulin inhibitors have been shown to be effective inhibitors of angiogenesis. We hypothesized that colchicine, a well-know tubulin inhibitor and 2-methoxyestradiol (2 MeOH), a novel tubulin inhibitor, would limit the initiation of a human angiogenic response and would limit subsequent neovessel growth in a dose-dependent manner. METHODS: To test this hypothesis, we cultured full-thickness human placental vein discs from three placentas in a fibrin-thrombin clot model. Both colchicine and 2 MeOH were tested over a wide range of concentrations (10(-6) to 10(-12) M) to determine their effect on the percent of wells that initiated an angiogenic response (%I) and the subsequent growth (Angiogenic Index, 0-16 range) of vein-derived neovessels. RESULTS: Colchicine at doses of 10(-6) and 10(-8) M completely inhibited the angiogenic response (CI: 95%, P < 0.0001) but lower (10(-10) to 10(-12) M) doses did not significantly inhibit angiogenesis (P = NS). Effective in vitro colchicine levels far exceed achievable non-toxic human plasma levels. In contrast, 2-methoxyestradiol decreased initiation and angiogenic growth significantly at 10(-6) M (CI: 95%, P < 0.0001), but did not significantly decrease angiogenesis at doses of 10(-8), 10(-10), or 10(-12) M. In contrast to colchicine, human plasma levels of 10(-6) M 2 MeOH are achievable clinically with little or no associated toxicity. CONCLUSIONS: Effective in vitro drug levels of 2 MeOH can be achieved in vivo, suggesting that 2 MeOH may have a role in the clinical treatment of angiogenesis-dependent diseases.

Actions of extracellular matrix on Sertoli cell morphology and function.

Sertoli cells were isolated and cultured in the absence or presence of extracellular matrix (ECM) to determine whether ECM may influence Sertoli cell function on a molecular level. As previously described, a morphological analysis of the cells indicated that ECM allows the expression of a columnar histotype and the formation of junctional complexes. The combined actions of ECM and hormones were found to have a profound effect in promoting the expression of a polarized Sertoli cell morphology. In our investigation of the effects of ECM on Sertoli cells, we used transferrin and androgen-binding protein (ABP) production as biochemical markers of Sertoli cell function. The presence of ECM was found to cause a 25% increase in the basal level of transferrin production; however, ECM had no effect on the basal level of ABP production by Sertoli cells. Regulatory agents such as follicle-stimulating hormone (FSH) and a combination of FSH, insulin, retinol, and testosterone stimulated the production of both transferrin and ABP. The ability of hormones to stimulate these Sertoli cell functions was not influenced by the presence of ECM. Similar results were obtained with 2-microns- or 50-microns-thick ECM and with a seminiferous tubule biomatrix preparation. ECM was found to increase the maintenance of long-term Sertoli cell cultures; however, the decline in Sertoli cell functional integrity, which occurs during cell culture, was not affected by the presence of ECM. An additional functional parameter examined was the radiolabeled proteins secreted by Sertoli cells. ECM did not promote the production or affect the electrophoretic profile of Sertoli cell-secreted proteins under basal or hormonally stimulated conditions. Combined results indicated that although ECM allowed the expression of a normal Sertoli cell histotype, ECM had no major effects on the Sertoli cell functions analyzed nor on the hormonal regulation of these functions. The inability of ECM to affect Sertoli cell function on a molecular level is discussed with regard to environmental as opposed to regulatory cellular interactions. Our observations imply that dramatic effects of ECM on cell morphology do not necessarily correlate to subsequent effects on cellular function.

Cytochemical and biochemical characterization of testicular peritubular myoid cells.

Testicular peritubular myoid cells secrete a paracrine factor that is a potent modulator of Sertoli cell functions involved in the maintenance of spermatogenesis. These cells also play an integral role in maintaining the structural integrity of the seminiferous tubule. To better understand this important testicular cell type, studies were initiated to characterize cultured peritubular cells using biochemical and histochemical techniques. The electrophoretic pattern of radiolabeled secreted proteins was similar for primary and subcultured peritubular cells and was unique from that of Sertoli cells. Morphologic differences between Sertoli cells and peritubular cells were noted and extended with histochemical staining techniques. Desmin cytoskeletal filaments were demonstrated immunocytochemically in peritubular cells, both in culture and in tissue sections, but were not detected in Sertoli cells. Desmin is proposed to be a marker for peritubular cell differentiation as well as a marker for peritubular cell contamination in Sertoli cell cultures. Peritubular cells and Sertoli cells were also stained histochemically for the presence of alkaline phosphatase. Staining for the alkaline phosphatase enzyme was associated with peritubular cells but not with Sertoli cells. Alkaline phosphatase is therefore an additional histochemical marker for peritubular cells. Biochemical characterization of peritubular cells relied on cell-specific enzymatic activities. Creatine phosphokinase activity, a marker for contractile cells, was found to be associated with peritubular cells, while negligible activity was associated with Sertoli cells. Alkaline phosphatase activity assayed spectrophotometrically was found to be a useful biochemical marker for peritubular cell function and was utilized to determine the responsiveness of primary and subcultured cells to regulatory agents. Testosterone stimulated alkaline phosphatase activity associated with primary cultures of peritubular cells, thus supporting the observation that peritubular cells provide a site of androgen action in the testis. Retinol increased alkaline phosphatase activity in subcultured peritubular cells. Alkaline phosphatase activity increased in response to dibutyryl cyclic adenosine monophosphate (AMP) in both primary and subcultured peritubular cell cultures. Observations indicate that the ability of androgens and retinoids to regulate testicular function may be mediated, in part, through their effects on peritubular cells. This provides additional support for the proposal that the mesenchymal-epithelial cell interactions between peritubular cells and Sertoli cells are important for the maintenance and control of testicular function. Results imply that the endocrine regulation of tissue function may be mediated in part through alterations in mesenchymal-epithelial cell interactions.

Purification of a paracrine factor, P-Mod-S, produced by testicular peritubular cells that modulates Sertoli cell function.

A testicular paracrine factor, P-Mod-S, was purified from conditioned medium obtained from serum-free cultures of peritubular cells. Stimulation of testicular transferrin production by cultured Sertoli cells was utilized as a bio-assay for P-Mod-S. A bioactive protein with an apparent molecular weight of 50,000 under physiological conditions was isolated by high pressure size exclusion chromatography. P-Mod-S was found to have an affinity for heparin and bound to a heparin affinity column. Two forms of P-Mod-S were purified with reverse-phase chromatography. The less hydrophobic form was referred to as P-Mod-S (A) and is a 56,000 molecular weight protein. The more hydrophobic form was referred to as P-Mod-S (B) and is a 59,000 molecular weight protein. Purification of P-Mod-S (A) and P-Mod-S (B) from peritubular cell-radiolabeled secreted proteins revealed that both proteins contain radioactivity. This result demonstrates active synthesis and secretion of P-Mod-S by peritubular cells. Although the amino acid composition of the two proteins indicates distinct differences in the content of several amino acids, the relationship of P-Mod-S (A) and P-Mod-S (B) is unknown at present. A greater than 1000-fold increase in the specific activity of P-Mod-S was achieved with the purification procedure utilized. P-Mod-S can account for essentially all the bioactivity present in crude peritubular cell-secreted protein preparations. The effects of the two forms of P-Mod-S on both transferrin and androgen-binding protein production by Sertoli cells was examined. Purified forms of P-Mod-S were found to have a greater effect on Sertoli cell function than any individual regulatory agent previously known to influence the cell, including follicle-stimulating hormone. The significance of peritubular cell-Sertoli cell interactions mediated via P-Mod-S to spermatogenesis and testicular function is discussed, as well as insight provided into general mesenchymal-epithelial cell interactions.

Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in normal and malignant human prostate.

BACKGROUND: Prostate cancer eventually becomes androgen-independent, suggesting that growth factors such as TGF beta 1-3 may potentially contribute to prostate neoplasia. The pattern and level of TGF beta 1-3 protein expression in normal and malignant human prostate are unknown. METHODS: An immunohistochemical study was undertaken to analyze TGF beta 1, TGF beta 2, and TGF beta 3 protein in malignant and adjacent normal prostates from 25 patients who had clinically localized prostate cancer. RESULTS: Normal prostate exhibited similar TGF beta 1 immunostaining in stromal and epithelial cells, whereas TGF beta 2 and TGF beta 3 protein staining was greater in the epithelial relative to the stromal compartments. In malignancy, prostate epithelial cells had higher TGF beta 1 and TGF beta 2 immunostaining than either the surrounding stromal cells or their normal prostatic epithelial counterparts. Although TGF beta 3 staining intensity was similar for both malignant and normal prostate epithelial cells, the pattern of staining switched from uniform apical to diffuse protein staining in malignant prostate glands. CONCLUSIONS: Prostate cancer was associated with alterations of TGF beta 1, TGF beta 2, and TGF beta 3 expression by prostatic epithelial cells which may play a role in prostatic carcinogenesis.