The tip-top branching ureter.

Organ rudiments with their epithelial bud and adjacent mesenchyme look much the same at their initial stage of differentiation. The subsequent branching of the epithelial anlagen determines the final pattern of the organs, but the mesenchyme provides essential signals for epithelial differentiation. Glial cell line derived neurotrophic factor (GDNF) has recently been shown to regulate ureteric branching morphogenesis and is thereby the first defined signalling molecule in the embryonic metanephric kidney. GDNF is expressed by the mesenchyme, binds to the tip of the ureteric bud and functions in both bud induction and bud orientation. The active receptor complex for GDNF includes the receptor tyrosine kinase Ret and a novel class of glycosylphosphatidylinositol-linked receptors, called GDNF family receptor alpha s.

Bmi-1 expression predicts prognosis in squamous cell carcinoma of the tongue.

BACKGROUND: The prognosis of squamous cell carcinoma of the oral tongue is poor and it would be beneficial to find prognostic markers to better adjust treatment. Bmi-1 controls cell cycle and self-renewal of tissue stem cells, transcription factor c-myc affects cell proliferation and apoptosis, and Snail regulates epithelial-mesenchymal transition. The expression of these markers has been connected to prognosis in many cancer types. METHODS: Bmi-1, c-myc, and Snail expressions were studied in our material consisting of 73 primarily T1N0M0 oral tongue carcinoma patients. We compared the immunoexpressions of Bmi-1, c-myc, and Snail with clinical parameters including the degree of histological differentiation, tumour size, TNM classification, depth of invasion, and resection margins. In addition, survival analyses were performed, comparing disease-free survival time with the registered protein expression of the markers mentioned above. RESULTS: A significant correlation between Bmi-1 protein expression and recurrence (log-rank test, P=0.005) was detected. Snail and c-myc expression did not correlate with prognosis. Snail expression correlated with histopathological grade (Fisher's exact test, P=0.007) and with the invasion depth of tumours (chi(2)-test, P=0.037). CONCLUSION: Negative Bmi-1 immunoexpression might serve as a marker of poor prognosis in oral tongue carcinoma patients.

Cellular origin of fibronectin in interspecies hybrid kidneys.

The cellular origin of fibronectin in the kidney was studied in three experimental models. Immunohistochemical techniques that use cross-reacting or species-specific antibodies against mouse or chicken fibronectin were employed. In the first model studied, initially avascular mouse kidneys cultured on avian chorioallantoic membranes differentiate into epithelial kidney tubules and become vascularized by chorioallantoic vessels. Subsequently, hybrid glomeruli composed of mouse podocytes and avian endothelial-mesangial cells form. In immunohistochemical studies, cross-reacting antibodies to fibronectin stained vascular walls, tubular basement membranes, interstitium, and glomeruli of mouse kidney grafts. The species-specific antibodies reacting only with mouse fibronectin stained interstitial areas and tubular basement membranes, but showed no reaction with hybrid glomeruli and avian vascular walls. In contrast, species-specific antibodies against chicken fibronectin stained both the interstitial areas and the vascular walls as well as the endothelial-mesangial areas of the hybrid glomeruli, but did not stain the mouse-derived epithelial structures of the kidneys. In the second model, embryonic kidneys cultured under avascular conditions in vitro develop glomerular tufts, which are devoid of endothelial cells. These explants showed fluorescence staining for fibronectin only in tubular basement membranes and in interstitium. The avascular, purely epithelial glomerular bodies remained unstained. Finally, in outgrowths of separated embryonic glomeruli, the cross-reacting fibronectin antibodies revealed two populations of cells: one devoid of fibronectin and another expressing fibronectin in strong fibrillar and granular patterns. These results favor the idea that the main endogenous cellular sources for fibronectin in the embryonic kidney are the interstitial and vascular cells. All experiments presented here suggest that fibronectin is not synthesized by glomerular epithelial cells in vivo.

Human laminin M chain (merosin): complete primary structure, chromosomal assignment, and expression of the M and A chain in human fetal tissues.

The primary structure of the human laminin M chain was determined from cDNA clones isolated from human placental libraries. The clones covered a total of 6,942 bp, with 49-bp encoding a 5' end untranslated region and 6,893-bp coding for a translated sequence. The complete human laminin M chain contains a 22-residue signal peptide and 3,088 residues of the mature M chain. The M chain has a domain structure similar to that of the human and mouse A chains. The homology between the two human laminin heavy chains is highest in the short arm region and lowest in the long arm helical domain I + II. Northern blot analysis of human fetal tissues showed that the M chain was expressed in most tissues such as cardiac muscle, pancreas, lung, spleen, kidney, adrenal gland, skin, testis, meninges, choroid plexus, and some other regions of the brain, but not in liver, thymus, and bone. In situ hybridization localized the expression of the M chain gene to cells of mesenchymal origin. In contrast, expression of the A chain was observed only in kidney, testis, neuroretina and some region of brain as determined by Northern analyses. Epithelial and endothelial cells were negative for both M and A chain gene transcripts. The gene for the human M chain (LAMM) was localized to chromosome 6q22-->23.

A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment.

We describe the identification of a novel laminin chain. Overlapping clones were isolated from a human fibrosarcoma HT1080 cell cDNA library spanning a total of 5,200 bp. A second set of clones contained an alternative 3' end sequence giving a total of 4,316 bp. The longer sequence contained an open reading frame for a 1,193-residue-long polypeptide. The alternative sequence was shortened at the carboxyl-terminal end coding for a 1,111-residue-long polypeptide. The amino acid sequence contained 21 amino acids of a putative signal peptide and 1,172 residues or alternatively 1,090 residues of a sequence with five distinct domains homologous to domains I-V in laminin chains. Comparison of the amino acid sequences showed that the novel laminin chain is homologous to the laminin B2 chain. However, the structure of the novel laminin chain isolated here differs significantly from that of the B2 chain in that it has no domain VI and domains V, IV, and III are shorter, resulting in a truncated laminin chain. The alternative sequence had a shortened domain I/II. In accordance with the current nomenclature, the chain characterized here is termed B2t. Calculation of possible chain interactions of laminin chains with the B2t chain domain I/II indicated that the B2t chain can replace the B2 chain in some laminin molecules. The gene for the laminin B2t chain (LAMB2T) was localized to chromosome 1q25-q31 in close proximity to the laminin B2 chain gene. Northern analysis showed that the B2t chain is expressed in several human fetal tissues but differently from the laminin B1 and B2 chains. By in situ hybridization expression of the B2t chain was localized to specific epithelial cells in skin, lung, and kidney as opposed to a general epithelial and endothelial cell expression of the laminin B2 chain in the same tissues.

Dependence of kidney morphogenesis on the expression of nerve growth factor receptor.

Nerve growth factor receptor (NGFR) serves as the binding site for the neurotrophic growth factors. Although NGFR has been found in several embryonic tissues outside the nervous system, the function of NGFR in embryogenesis of non-neuronal organs remains unknown. NGFR is transiently synthesized by embryonic rat kidney and disappears from nephrons upon their terminal differentiation. Anti-sense oligonucleotide inhibition of NGFR expression inhibits kidney morphogenesis. Therefore, NGFR is required not only for development of the nervous system, but also for differentiation of the kidney tubules.

Regulation of cell fate decision of undifferentiated spermatogonia by GDNF.

The molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Transgenic loss-of-function and overexpression models now show that the dosage of glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, regulates cell fate decisions of undifferentiated spermatogonial cells that include the stem cells for spermatogenesis. Gene-targeted mice with one GDNF-null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. They are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. Nonmetastatic testicular tumors are regularly formed in older GDNF-overexpressing mice. Thus, GDNF contributes to paracrine regulation of spermatogonial self-renewal and differentiation.

Stem cell protein BMI-1 is an independent marker for poor prognosis in oligodendroglial tumours.

AIMS: The polycomb factor BMI-1 has recently been implicated in tumorigenesis of the central nervous system in several experimental animal models. However, the significance of BMI-1 in human glioma has not been investigated. Here we describe expression of the polycomb protein BMI-1 and its downstream targets p16(Ink4a) and MDM2 in both high- and low-grade human glioma. METHODS: Tumour samples were collected from 305 adult patients treated for primary grades 2-4 gliomas between 1980 and 2006 in Finland and Germany. BMI-1, p16 and MDM2 expression was evaluated using immunohistochemistry in representative paraffin-embedded tumour tissue. The significance of observed immunoreactivity, age at onset, gender, histopathological findings and proliferative index was analysed in univariate and multivariate survival models. RESULTS: BMI-1 was expressed in all histologic types of diffuse gliomas. We found a significant correlation (P = 0.007) between the frequency of BMI-1 immunoreactive tumour cells and poor survival in World Health Organization grades II-III oligodendrogliomas and oligoastrocytomas (n = 62). The median survival of patients grouped by low, intermediate or high frequency of BMI-1 immunoreactive tumour cells was 191 months, 151 months and 68 months, respectively. This association was also significant in the Cox multivariate regression model. Nuclear p16 immunopositivity predicted better survival in astrocytomas and an inverse correlation between p16 expression and the Ki-67 mitotic index was also observed. CONCLUSIONS: BMI-1 is found in all histological types of gliomas and the relative protein expression of BMI-1 is a novel independent prognostic marker in oligodendroglial tumours.

E6/E7 oncogenes increase and tumor suppressors decrease the proportion of self-renewing neural progenitor cells.

Many if not most tissues need a controlled number of stem cells to maintain normal function. Cancer can be seen as a process of disturbed tissue homeostasis, in which too many cells have or acquire too primitive identity. Here we measured how oncogenes and tumour suppressors affect the differentiation capacity, proportion and characteristics of progenitor cells in a model tissue. Neural progenitor cells (NPCs) were exposed to human papilloma virus E6, E7 or E6/E7 oncogenes, which degrade tumour suppressors p53 and pRb family members, respectively. E6/E7-expressing or p53-/- NPCs were able to differentiate, but simultaneously retained high capacity for self-renewal, proliferation, ability to remain multipotent in conditions promoting differentiation and showed delayed cell cycle exit. These functions were mediated through p53 and pRb family, and involved MEK-ERK signalling. Decreased amount of p53 increased self-renewal and proliferation, whereas pRb affected only proliferation. Our results suggest that the oncogenes increase whereas p53 and pRb family tumour suppressors decrease the number and proportion of progenitor cells. These findings provide one explanation how oncogenes and tumour suppressors control tissue homeostasis and highlight their importance in stem cell self- renewal, linked both to cancer and life-long tissue turnover.

Integrins and laminins in human renal carcinoma cells and tumors grown in nude mice.

We studied by indirect immunofluorescence microscopy the distribution of integrins and laminins in four human renal cell carcinoma cell lines (CAKI-2, A498, CAKI-1, and ACHN) in vitro and in s.c. xenografts in nude mice. In vitro, all four cell lines expressed the alpha 1, alpha 3, alpha v, beta 1, beta 3, and beta 5 subunits and three expressed the alpha 6 subunit; all cell lines expressed laminin A, B1, and B2 chains. Histologically, the CAKI-2 and A498 cells formed differentiated grade 1 (G1) and G2 tumors, respectively, while the CAKI-1 and ACHN cells formed poorly differentiated G3 tumors. The described integrin profile was largely retained upon xenografting. Basal polarization of the alpha 3 and alpha 6 integrin subunits was found in the differentiated tumors, and human laminins were detected as discrete linear structures surrounding tumor cell clusters in these tumors, suggesting that the cells have retained a polarized cell-laminin interaction characteristic of normal tubular epithelial cells. A disorganized distribution of integrins and laminins was noted in the G3 tumors. We conclude that these renal carcinoma cell lines displayed an integrin repertoire similar to that of clinical renal carcinomas and retained it upon xenografting. Furthermore, the organization of integrins and laminins in the xenografts correlated with histological grade.

Promotion of seminomatous tumors by targeted overexpression of glial cell line-derived neurotrophic factor in mouse testis.

We show with transgenic mice that targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) in undifferentiated spermatogonia promotes malignant testicular tumors, which express germ-cell markers. The tumors are invasive and contain aneuploid cells, but no distant metastases have been found. By several histological, molecular, and histochemical characteristics, the GDNF-induced tumors mimic classic seminomas in men, representing a useful experimental model for testicular germ-cell tumors. The data also show that a deregulated stimulation of a normal proto-oncogene by its ligand can be an initiative event in carcinogenesis.

Percutaneous needle biopsy preceding preoperative chemotherapy in the management of massive renal tumors in children.

While the National Wilms' Tumor Study (NWTS) Group in the United States puts an emphasis on accurate staging and histology before any therapy is given for Wilms' tumor, the International Society of Pediatric Oncology (SIOP) in Europe focuses on preoperative therapy and safer surgery. Our current approach combines the benefits of both policies in the management of massive renal tumors in children. In seven consecutive patients we first obtained a percutaneous posterior needle biopsy to obtain adequate tissue for histology, and proceeded with preoperative chemotherapy with vincristine and dactinomycin until tumor shrinkage was sufficient. Tumor removals were feasible and uneventful. At the time of operation, two tumors were found to be totally or almost totally necrotic. In the others, which still included viable tumor, the histology corresponded well to the needle biopsy findings. One case with unfavorable histology and one with rhabdoid sarcoma would have been missed and given suboptimal therapy without the primary needle biopsy. As possible biopsy-related complications, subcapsular intratumoral bleeding was recognized in two patients. We conclude that percutaneous posterior needle biopsy is safe and yields definite, detailed histology in massive renal tumors in children. Preoperative chemotherapy facilitates surgery in these patients.

In vivo purging of bone marrow in children with poor-risk neuroblastoma for marrow collection and autologous bone marrow transplantation.

PURPOSE: To evaluate the following prospectively in poor-risk neuroblastoma (NBL) patients: (1) the feasibility and efficacy of in vivo purging of bone marrow; and (2) the outcome after autologous bone marrow transplantation (ABMT) when immunologically tumor-free, unpurged autografts were used. PATIENTS AND METHODS: Twenty-three children with poor-risk NBL were evaluated during induction chemotherapy by repeat bone marrow examinations, including aspirate, biopsy, and an immunofluorescence method using the anti-GD2 monoclonal antibody 3A7. Nineteen patients completed the program with surgery with or without local irradiation followed by ABMT. RESULTS: Autologous bone marrow grafts, both immunologically and cytologically clean, were obtained and used in 19 of 23 children. The overall 4-year disease-free survival of the 19 grafted children was 53%, with a toxic death rate of 16% and a posttransplant relapse rate of 37%. According to the in vivo purging efficacy of the 18 children with initial marrow disease, the following three groups were formed: patients with (1) perfect in vivo purging (n = 5); (2) eventually successful in vivo purging (n = 8); and (3) unsuccesful in vivo purging (n = 5). The 4-year DFS was 100%, 67%, and 0%, respectively (P < 0.001). The five patients with unsuccessful in vivo purging failed because of resistant/progressive bulky disease. CONCLUSION: In patients with poor-risk NBL, in vivo purging of bone marrow by conventional chemotherapy is feasible, can be monitored, and the purging efficacy during the first 3 months after diagnosis is a strong prognostic factor reflecting tumor responsiveness to therapy. Autografting with immunologically clean, unpurged marrows gives a DFS well comparable to previous studies using ex vivo purging.

GDNF and its receptors in the regulation of the ureteric branching.

Recent transgenic and organ culture experiments have inevitably shown that glial cell line-derived neurotrophic factor (GDNF) is a mesenchyme-derived signal for ureteric budding and branching. The signalling receptor complex for GDNF includes a dimer of Ret receptor tyrosine kinase and two molecules of GDNF family receptor alpha1. Alpha-receptors are not only needed for the ligand binding and Ret activation, but they might mediate signals without Ret. While GDNF is clearly required for ureteric branching, tissue recombination studies have shown that it is not sufficient for the completion of ureteric morphogenesis, and other signalling molecules are needed. Different experimental models have resulted in somewhat contradictory results on their molecular identity, but transforming growth factor-beta1, -beta2, fibroblast growth factor-7 and hepatocyte growth factor form, obviously among others, a redundant set of growth factors in ureteric differentiation. Three other members of the GDNF family, neurturin, artemin and persephin, are also expressed in the developing kidney, and at least neurturin and persephin promote ureteric branching in vitro, but their true in vivo roles are still unclear.

The effect of neuronal cells on kidney differentiation.

During embryonic growth, tissue interactions between dissimilar cells are the driving forces of morphogenesis. Although their importance has been well known for over the past 50 years, the molecular background of these interactions has remained unelucidated. The unrecognized heterogeneity of those mesenchymal cells that are involved in the epithelio-mesenchymal tissue interactions may be one reason for this. For example, studies of kidney differentiation show that the metanephric organ rudiment contains more cell-lines than previously thought. Identification of both neural crest- and mesoderm-derived cells in the nephrogenic mesenchyme helps in re-evaluating the biology of the tubule induction. The neural crest-derived cells of the nephric rudiment differentiate into neuronal cells, and later during differentiation some of them are found in the stroma. There is also experimental evidence for the role of these neuronal cells in the morphogenetic tissue interaction.

Neuronal characteristics in embryonic renal stroma.

The metanephric mesenchyme is considered a homogeneous population of predetermined, but pluripotent cells with a nephrogenic bias. By an inductive stimulus, the mesenchyme is programmed to differentiate into the various epithelial phenotypes of the secretory nephron. A fraction of the mesenchymal cells, however, remains in the interstitium between the nephrons and differentiates into spindle-shaped, clear-cytoplasmic renal stroma. We have analyzed the molecular nature of these cells in order to discover the specific cell types that could be involved in the morphogenetic processes during kidney differentiation. In situ hybridization reveals neurofilament light protein mRNA, and immunohistology shows neurofilament light and medium proteins in the stromal cells around kidney tubules. By immunohistochemistry these peritubular stromal cells can be distinguished from the neuronal cells of the renal microganglion: the peritubular stromal cells are neurofilament-positive but L1 neural cell adhesion protein-negative, whereas the neuronal cells with axonal extension are both neurofilament-positive and L1 neural cell adhesion protein-positive. Proliferation index of the stromal cells was low as compared to tubular cells, as shown by bromodeoxyuridine incorporation.

The human laminin beta 2 chain (S-laminin): structure, expression in fetal tissues and chromosomal assignment of the LAMB2 gene.

The sequence of the human laminin beta 2 chain (previously s-laminin) was derived from cloned cDNAs. The complete translation product has 1798 amino acid residues, including a 32-residue signal peptide. The human chain lacks the tripeptide sequence LRE in domain I which is present in the rat polypeptide chain and has been shown to promote motor neuronal cell adhesion. The human gene (LAMB2) was localized to chromosome 3p21 using somatic cell hybrids and fluorescent in situ hybridization analysis. Northern and in situ hybridization analyses from numerous fetal tissues revealed that the beta 2 chain is generally widely expressed. beta 2, but not beta 1, was shown by in situ hybridization to be expressed in fetal brain and renal glomeruli. In fetal skin, beta 2 was expressed both in epidermal and dermal cells, while beta 1 was expressed only in the dermis. Expression of beta 2 in fetal liver was seen in hepatocytes, while no signals were observed for beta 1. In lung, both beta 1 and beta 2 were expressed in alveoli and bronchial smooth muscle cells, whereas only the beta 2 chain was expressed in bronchial epithelial cells. In striated muscle, however, the beta 1 chain, but not beta 2, was expressed. These results indicate different biological roles for the laminin beta 1 and beta 2 chains.

Primary structure and expression of a novel human laminin alpha 4 chain.

The complete primary structure of a novel human laminin alpha 4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature alpha 4 chain of 1792 residues. The domain structure is similar to that of the recently described alpha 3 chain [Ryan, Tizard, Van Devanter and Carter (1994) J. Biol. Chem. 269, 22779-22787]. Northern analysis of RNA from human fetal and adult tissues revealed developmental regulation of expression. In adult, strong expression was observed in heart as well as lung, ovary, small and large intestines, placenta and liver, whereas weak or no expression was detected in skeletal muscle, kidney, pancreas, testis, prostate or brain. In contrast, fetal lung and kidney revealed high expression. In situ hybridization analysis of human fetal and newborn tissues showed expression of the laminin in alpha 4 chain in certain mesenchymal cells in tissues such as smooth muscle and dermis.

Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides.

We have identified a synthetic peptide derived from the B2-chain of mouse laminin, Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile (p20), which stimulates the neurite outgrowth-promoting activity of the native molecule. In organotypic cultures, neurons from newborn mouse brain or embryonic peripheral nervous system responded by extensive neurite outgrowth for native laminin or the peptide p20 in the culture medium. If rat cerebellar neurons were grown on laminin, 1-5 microM (1-5 micrograms/ml) of peptide p20 in the culture medium competed with laminin and inhibited neuronal attachment and neurite outgrowth, whereas higher concentrations (greater than 50 microM; greater than 50 micrograms/ml) had a specific neurotoxic effect. When peptide p20 was used as the culture substratum, neurite outgrowth in cerebellar cultures was up to 60% of that seen on native laminin. Our results indicate that a neurite outgrowth-promoting domain of laminin is located in the alpha-helical region of the B2-chain, and is active for both central and peripheral neurons.