Assessment of fresh and cryopreserved testicular tissues from (pre)pubertal boys during organ culture as a strategy for in vitro spermatogenesis.

STUDY QUESTION: Can the organ culture method be applied to both fresh and cryopreserved human (pre)pubertal testicular tissue as a strategy for in vitro spermatogenesis? SUMMARY ANSWER: Although induction of spermatogenesis was not achieved in vitro, testicular architecture, endocrine function and spermatogonial proliferation were maintained in both fresh and cryopreserved testicular tissues. WHAT IS KNOWN ALREADY: Cryopreservation of a testicular biopsy is increasingly offered as a fertility preservation strategy for prepubertal cancer patients. One of the proposed experimental approaches to restore fertility is the organ culture method, which, in the mouse model, successfully allows for in vitro development of spermatozoa from testicular biopsies. However, complete spermatogenesis from human prepubertal testicular tissue in such an organ culture system has not been demonstrated. STUDY DESIGN, SIZE, DURATION: Testicular tissue was collected from nine (pre)pubertal boys diagnosed with cancer (ranging from 6 to 14 years of age) admitted for fertility preservation before treatment. Testicular biopsies were either immediately processed for culture or first cryopreserved, using a controlled slow freezing protocol, and thawed before culture. Organ culture of testicular fragments was performed in two different media for a maximum period of 5 weeks, targeting early cellular events (viability, meiosis and somatic differentiation) in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fresh and cryopreserved-thawed testis fragments (1-2 mm3) were cultured at a gas-liquid interphase (34°C, 5% CO2) in Minimum Essential Medium alpha + 10% knock-out serum replacement medium containing 10-7 M melatonin and 10-6 M retinoic acid, with or without 3 IU/L FSH/LH supplementation. The effect of culture conditions on testicular fragments was weekly assessed by histological evaluation of germ cell development and immunohistochemical identification of spermatogonia (using MAGEA4), proliferative status of spermatogonia and Sertoli cells (using proliferating cell nuclear antigen [PCNA]), intratubular cell apoptosis (by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) and Sertoli cells maturation (using Anti-Müllerian Hormone [AMH] versus Androgen Receptor [AR]). Additionally, Leydig cells' functionality was determined by measuring testosterone concentration in the culture media supernatants. MAIN RESULTS AND THE ROLE OF CHANCE: Neither fresh nor cryopreserved human (pre)pubertal testicular fragments were able to initiate spermatogenesis in our organ culture system. Nonetheless, our data suggest that fresh and cryopreserved testicular fragments have comparable functionality in the described organ culture conditions, as reflected by the absence of significant differences in any of the weekly evaluated functional parameters. Additionally, no significant differences were found between the two tested media when culturing fresh and cryopreserved human testicular fragments. Although spermatogonia survived and remained proliferative in all culture conditions, a significant reduction of the spermatogonial population (P ≤ 0.001) was observed over the culture period, justified by a combined reduction of proliferation activity (P ≤ 0.001) and increased intratubular cell apoptosis (P ≤ 0.001). We observed a transient increase in Sertoli cell proliferative activity, loss of AMH expression (P ≤ 0.001) but no induction of AR expression. Leydig cell endocrine function was successfully stimulated in vitro as indicated by increased testosterone production in all conditions throughout the entire culture period (P ≤ 0.02). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although not noticeable in this study, we cannot exclude that if an optimized culture method ensuring complete spermatogenesis in human testicular fragments is established, differences in functional or spermatogenic efficiency between fresh and cryopreserved tissue might be found. WIDER IMPLICATIONS OF THE FINDINGS: The current inability to initiate spermatogenesis in vitro from cryopreserved human testicular fragments should be included in the counselling of patients who are offered testicular tissue cryopreservation to preserve fertility. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by EU-FP7-PEOPLE-2013-ITN 603568 `Growsperm'. None of the authors have competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Development of the testis in pre-pubertal boys with cancer after biopsy for fertility preservation.

STUDY QUESTION: Is testicular growth affected by a testicular biopsy intended for fertility preservation in pre-pubertal boys with cancer? SUMMARY ANSWER: Testicular growth of the biopsied testis is not impeded in comparison to the non-biopsied contralateral testis up until 1 year after surgery. WHAT IS KNOWN ALREADY: Fertility preservation in pre-pubertal boys by means of testicular biopsy has been conducted for more than 15 years. Although immediate adverse effects of testicular biopsy are rare (1%), no data exist on the effect of biopsy on testicular growth. STUDY DESIGN, SIZE, DURATION: In this prospective cohort study, between March 2011 and February 2017, 93 parents of pre-pubertal boys were offered cryopreservation of testicular tissue of their son, of whom 78 consented. Sixty-four boys were included in this follow-up study. PARTICIPANTS/MATERIALS, SETTING, METHODS: All boys with cancer at the paediatric oncology department of the Academic Medical Center (AMC) who needed gonadotoxic therapy and were unable to ejaculate were offered cryopreservation of testicular tissue prior to treatment. By testicular ultrasound before and after biopsy (1, 6 and 12 months after biopsy), volume and parenchymal abnormalities were assessed. Data were analysed using mixed-effects modelling. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 64 included boys all were followed up at 1 month, 58 at 6 months and 55 at 12 months. Mean testicular volumes after 1, 6 and 12 months after biopsy were 1.7 ± 2.1, 1.7 ± 2.2 and 1.9 ± 2.4 for the biopsied testis and 1.8 ± 2.2, 1.8 ± 2.3 and 2.0 ± 2.2 for the non-biopsied testis, respectively. Biopsy of the testis did not have a significant impact on testicular growth. Immediate adverse effects of the biopsy, i.e. wound infections, were seen in 3/78 boys (3.8%). LIMITATIONS, REASONS FOR CAUTION: Although it is the largest cohort available to date, the number of patients included in our follow-up is still relatively small. A larger cohort would be able to evaluate growth more precisely. Follow-up was discontinued in a significant portion of boys, 12/76 (15.8%), mainly because of death due to primary illness but also because they could not be reached or declined further follow-up. WIDER IMPLICATIONS OF THE FINDINGS: These reassuring data may be used in counselling future boys who are eligible for fertility preservation and their parents. STUDY FUNDING/COMPETING INTEREST(S): Study funded by KIKA Foundation (Kika 86), Grant from the Netherlands Organisation for Health Research and Development (ZonMW TAS-116003002). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: CCMO-register: NL27690.000.09.

c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route.

Cbl family members have an evolutionarily conserved role in attenuating receptor tyrosine kinase function. Their negative regulatory capacity depends on a Ring finger domain that interacts with ubiquitin conjugating enzymes. Cbl molecules constitute a novel type of E3 or ubiquitin ligase family that is recruited to phosphotyrosine motifs. Ubiquitination of the receptor system is coupled to its downregulation, but it is unclear at which point in the endocytic pathway Cbl molecules come into play. Using low temperature and a dynamin mutant, we find that c-Cbl associates with and ubiquitinates the activated epidermal growth factor (EGF) receptor at the plasma membrane in the absence of endocytosis. With the aid of confocal microscopy and immunogold electron microscopy, we could demonstrate that c-Cbl associates with the EGF receptor at the plasma membrane prior to receptor recruitment into clathrin-coated pits and remains associated throughout the clathrin-mediated endocytic pathway. c-Cbl and the EGF receptor also colocalize in internal vesicles of multivesicular endosomes. Our data are consistent with a role for c-Cbl in clathrin-mediated endocytosis of tyrosine kinase receptors, as well as their intracellular sorting.

Glomerular extracellular matrix components and integrins.

It has become apparent that extracellular matrix components and their cellular receptors, the integrins, are important regulators of glomerular development and function. In this rapidly evolving field we studied the production of extracellular matrix components and integrins by rat glomerular visceral epithelial and mesangial cells, using molecular probes and antibodies that have recently become available. Special attention was paid to laminin isoforms and to splice variants of the integrin subunits alpha 3 and alpha 6. Results were compared to the in vivo expression in human fetal, newborn and adult kidneys. The mesangial cells were found to produce laminin-1, nidogen and two as yet unidentified laminin isoforms with putative alpha chains of about 395 (alpha x) and of 375 kDa (alpha y), tentatively described before as bovine kidney laminin. Furthermore, they expressed the integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3A beta 1, alpha 5 beta 1, alpha v beta 3, alpha v beta 5, and small amounts of alpha 6A beta 1 and alpha 6B beta 1. The glomerular visceral epithelial cells produced the two new laminin isoforms mentioned above, laminin-5, but no laminin-1 or nidogen. The integrins alpha 2 beta 1, alpha 3A beta 1, alpha 6A beta 4, alpha 6B beta 4 and the integrin subunit alpha v were found to be expressed. We show that during nephrogenesis, the laminin alpha 1 chain disappears and is replaced by another alpha chain, possibly one of the two as yet unidentified alpha chains mentioned above. The laminin beta 1 chain is replaced by the beta 2 chain somewhat later in glomerular development. In general, the integrins found to be expressed in glomeruli of adult kidney were consistent with those found in cultured glomerular visceral epithelial and mesangial cells. No splice variant switch of the integrin alpha 3 or alpha 6 subunits could be demonstrated during nephrogenesis. Our results suggest an important role for the mesangial cell in providing nidogen as a crucial component of the supramolecular structure of the glomerular basement membrane. Furthermore our results indicate that laminin alpha x beta 2 gamma 1 and alpha y beta 2 gamma 1 isoforms are important in the glomerulus of adult kidney and that the integrin alpha 3A beta 1 is the main integrin receptor for laminin isoforms on glomerular visceral epithelial and mesangial cells, both in vitro and in vivo.

Identification of the cleavage sites in the alpha6A integrin subunit: structural requirements for cleavage and functional analysis of the uncleaved alpha6Abeta1 integrin.

The alpha6A and alpha6B integrin subunits are proteolytically cleaved during biosynthesis into a heavy chain (120 kDa) that is disulphide-linked to one of two light chains (31 or 30 kDa). Analysis of the structure of the alpha6A subunit on the carcinoma cell line T24 and human platelets demonstrated that the two light chains of alpha6 are not differentially glycosylated products of one polypeptide. Rather they possess different polypeptide backbones, which presumably result from proteolytic cleavage at distinct sites in the alpha6 precursor. Mutations were introduced in the codons for the R876KKR879, E883K884, R890K891 and R898K899 sequences, the potential proteolytic cleavage sites, and wild-type and mutant alpha6A cDNAs were transfected into K562 cells. The mutant alpha6A integrin subunits were expressed in association with endogenous beta1 at levels comparable to that of wild-type alpha6Abeta1. A single alpha6 polypeptide chain (150 kDa) was precipitated from transfectants expressing alpha6A with mutations or deletions in the RKKR sequence. Mutations in the EK sequence yielded alpha6A subunits that were cleaved once into a heavy and a light chain, whereas alpha6A subunits with mutations in one of the two RK sequences were, like wild-type alpha6A, cleaved into one heavy and two light chains. Thus a change in the RKKR sequence prevents the cleavage of alpha6. The EK site is the secondary cleavage site, which is used only when the primary site (RKKR) is intact. Microsequencing of the N-termini of the two alpha6A light chains from platelets demonstrated that cleavage occurs after Arg879 and Lys884. Because alpha6(RKKG), alpha6(GKKR) and alpha6(RGGR) subunits were not cleaved it seems that both the arginine residues and the lysine residues are essential for cleavage of RKKR. alpha6A mutants with the RKKR sequence shifted to the EK site, in such a way that the position of the arginine residue after which cleavage occurs corresponds exactly to Lys884, were partly cleaved, whereas alpha6A mutants with the RKKR sequence shifted to other positions in the alpha6A subunit, including one in which it was shifted two residues farther than the EK cleavage site, were not cleaved. In addition, alpha6A mutants with an alpha5-like cleavage site, i.e. arginine, lysine and histidine residues at positions -1, -2 and -6, were not cleaved. Thus both an intact RKKR sequence and its proper position are essential. After activation by the anti-beta1 stimulatory monoclonal antibody TS2/16, both cleaved and uncleaved alpha6Abeta1 integrins bound to laminin-1. The phorbol ester PMA, which activates cleaved wild-type and mutant alpha6Abeta1, did not activate uncleaved alpha6Abeta1. Thus uncleaved alpha6Abeta1 is capable of ligand binding, but not of inside-out signalling. Our results suggest that cleavage of alpha6 is required to generate a proper conformation that enables the affinity modulation of the alpha6Abeta1 receptor by PMA.

The A and B variants of the alpha 3 integrin subunit: tissue distribution and functional characterization.

The alpha subunits of the laminin-binding integrins alpha 3 beta 1, alpha 6 beta 1, and alpha 7 beta 1 have homologous sequences and are similar in structure. Two cytoplasmic variants, A and B, have been identified for each of these alpha subunits, although the alpha 3B splice variant has been detected only at the mRNA level. We prepared a panel of mouse monoclonal antibodies specific for the A and B variants of the alpha 3 subunit to study their tissue distribution. Four monoclonal antibodies react with alpha 3A, one of which recognizes only the nonphosphorylated form; of the three anti-alpha 3B antibodies, one cross-reacts with alpha 6B. Reverse transcriptase-PCR analysis of various human tissues revealed the presence of alpha 3B mRNA in brain, heart, and skeletal muscle. Moreover, the alpha 3B protein was detected by immunoblotting in brain and heart tissue but not in skeletal muscle. In contrast, alpha 3A mRNA and protein were present in all tissues studied. Thus, the expression of alpha 3B in adult tissues is more restricted than that of alpha 3A. Immunohistochemical studies showed that in brain tissue, both variants are exclusively expressed on small blood-vessel endothelium, whereas in heart tissue their distribution patterns differ markedly. Although alpha 3A is strongly expressed on vascular smooth muscle cells, alpha 3B is detected only on endothelial cells of veins. Expression of the two variant forms of alpha 3 in K562 cells revealed that the ligand-binding specificities of alpha 3A beta 1 and alpha 3B beta 1 are identical: both bind human laminin-2 and -4, laminin-5, and laminins isolated from bovine kidney, but not bovine laminin-2 and -4, mouse laminin-1, or human fibronectin. In addition, adhesion mediated by both integrins is induced to the same extent by phorbol 12-myristate 13-acetate. The alpha 3A, but not the alpha 3B subunit, is phosphorylated; and phosphorylation of alpha 3A increases after phorbol 12-myristate 13-acetate stimulation. Thus, we found no differences between the adhesion functions of the A and B variants of alpha 3.

Distribution of laminin variants and their integrin receptors in human secondary lymphoid tissue. Colocalization suggests that the alpha 6 beta 4-integrin is a receptor for laminin-5 in lymphoid follicles.

Laminins are a family of multifunctional basement membrane glycoproteins. Over the last years, many laminin isoforms have been characterized, which were shown to be composed of distinct combinations of variant alpha, beta and gamma chains. Some of these isoforms show remarkable tissue specificity, which suggests functional involvement in local processes. In this study the previously described mAb 4C7, which recognize epithelial basement membranes as well as endothelial basement membranes in lymphoid follicles, was identified as an anti-laminin-5 antibody. Using a set of mAbs against various variant laminin chains we established that specifically the gamma 2 chain of laminin-5 was confined to the endothelial basement membranes of vessels in lymphoid follicles, whereas other variant laminin chains were also expressed elsewhere in the lymphoid follicles, whereas other variant laminin chains were also expressed elsewhere in the lymphoid tissue. Additionally, the expression of the known integrin receptors of laminin-5 was also examined. The alpha 6 beta 4 integrin-receptor for laminin was found to be colocalized with the laminin-5 gamma 2 chain on the abluminal surface of endothelial cells, whereas the alpha 3 integrin chain could not be detected in lymphoid follicles. This finding suggests that the alpha 6 beta 4 integrin (and not the alpha 3 beta 1 integrin) serves as a laminin-5 receptor on endothelial cells in the follicular compartment of lymphoid tissue. Furthermore, alpha 6 beta 4 was also found in the same punctuated pattern on FDCs as laminin-5. The function of the laminin-alpha 6 beta 4 complex in this particular localisation is still obscure, but a role in the maintainance of the follicular compartment via hemidesmosome-like attachment sites is postulated.

The role of the cytoplasmic domain of alpha 6 integrin in the assembly and function of alpha 6 beta 1 and alpha 6 beta 4.

We have studied the role of the cytoplasmic domain of alpha 6 in the assembly and function of the alpha 6 beta 4 integrin, and compared it with the role of alpha 6 in the assembly and function of alpha 6 beta 1, by transfection of cDNAs encoding cytoplasmic mutants of alpha 6 into K562 cells with or without full-length beta 4 cDNA. Des-(1022-1050)-alpha 6, which contains a deletion C-terminal to the GFFKR motif, was expressed in association with beta 1, but associated preferentially with beta 4, whereas the wild-type alpha 6 subunit associated efficiently with beta 1 and beta 4. Des-(1016-1050)-alpha 6, which lacked also the GFFKR sequence, was only expressed at the cell surface when beta 4 was available. Transient expression in COS-7 cells showed that des-(1016-1050)-alpha 6 was retained in the endoplasmic reticulum as a monomer, which suggests that truncation of the cytoplasmic domain reduces the affinity of alpha 6 for beta 1, particularly when the GFFKR sequence is absent. Although the GFFKR motif is not essential for association of alpha 6 with beta 4, it increases the stability of the alpha 6 beta 4 integrin. The cytoplasmic domain of alpha 6 is essential for inside-out and outside-in signaling via the alpha 6 beta 1 receptor, but not for adhesion via alpha 6 beta 4. We show that alpha 6 beta 4 is a constitutively active receptor. Thus, unlike adhesion by most other integrins, adhesion by alpha 6 beta 4 does not seem to depend on any active cellular process. Binding of alpha 6 beta 4 to ligand was only slightly affected by truncation of the alpha 6 cytoplasmic domain N-terminal to the GFFKR sequence and became partially dependent on metabolic energy. These data indicate that truncations of the cytoplasmic domain of the alpha 6 subunit affect the assembly and function of alpha 6 beta 1 more strongly than those of alpha 6 beta 4. This difference may be due to the greater affinity of alpha 6 for beta 4 than for beta 1, which makes alpha 6 beta 4 less susceptible to the effect of truncations.

The alpha 6 beta 4 integrin is a receptor for both laminin and kalinin.

Previously, we have establish K562 transfectants that express either alpha 6A beta 1 or alpha 6B beta 1 (K alpha 6A or K alpha 6B) on their surface. Both cell lines bind to laminin and kalinin after treatment with the beta 1-stimulatory antibody TS2/16. Here we introduce the full-length beta 4 cDNA into the alpha 6A- and alpha 6B-expressing K562 cells and selected stably transfected cells. The beta 4 subunit was expressed on the surface of both transfectants and it formed dimers with the alpha 6A or alpha 6B subunits. Immunoprecipitation and preclearing analyses revealed that both transfectants expressed alpha 6 beta 1, in addition to alpha 6 beta 4. While K alpha 6A and K alpha 6B cells required TS2/16 stimulation for binding to laminin or kalinin, adhesion of the unstimulated beta 4-transfected K alpha 6A and K alpha 6B cells to these matrix components was already substantial. This adhesion was mediated by both alpha 6 beta 1 and alpha 6 beta 4 since it was completely blocked by an alpha 6-specific antibody or by a combination of anti-beta 1 and anti-beta 4 antibodies, but only partially by either of these latter two antibodies alone. Adhesion to laminin was completely blocked by an antiserum to laminin fragment E8 as was the adhesion to kalinin by an antibody to kalinin, demonstrating the specificity of adhesion. Both transfectants always adhered more strongly to kalinin than to laminin. Furthermore, binding to kalinin was less well blocked by antibodies to beta 4 than binding to laminin, indicating that the affinity of alpha 6 beta 4 for kalinin is higher than that for laminin. The fact that alpha 6 beta 1 mediated adhesion without TS2/16 stimulation on the beta 4-transfected K alpha 6A and K alpha 6B cells suggests that some activation of alpha 6 beta 1 had occurred in these cells, even though binding was increased when they were actively stimulated by the antibody TS2/16. Finally, we show that Mn2+ induced binding of solubilized alpha 6 beta 4 to matrix containing kalinin, deposited by the murine cell line RAC-11P/SD. This binding was inhibited by the anti-alpha 6 mAb GoH3. Together, these results indicate that both alpha 6 beta 1 and alpha 6 beta 4 are receptors for laminin and kalinin and that there are no differences in ligand specificity between the A and B variants of the alpha 6 subunit when associated with either beta 1 or beta 4.

Distinct and overlapping ligand specificities of the alpha 3A beta 1 and alpha 6A beta 1 integrins: recognition of laminin isoforms.

The ligand specificity of the alpha 3A beta 1 integrin was analyzed using K562 cells transfected with full-length alpha 3A cDNA and was compared with that of alpha 6A beta 1 in similarly transfected K562 cells. Clones were obtained that showed comparable surface expression of either alpha 3A beta 1 or alpha 6A beta 1 integrins. Those expressing alpha 3A beta 1 attached to and spread on immunopurified human kalinin and cellular matrices containing human kalinin, which is a particular isoform of laminin. In addition, alpha 3A transfectants adhered to bovine kidney laminins possessing a novel A chain variant. Binding to kalinin was blocked by a monoclonal antibody against the A chain constituent of kalinin and adhesion to both kalinin and kidney laminins by anti-alpha 3 and beta 1 monoclonal antibodies. The alpha 3A transfected cells bound more strongly to kalinin and bovine kidney laminins after treatment with the beta 1 stimulatory antibody TS2/16. A distinctly weaker and activation-dependent adhesion of alpha 3A transfectants was observed on human placental laminins possessing the Am chain variant (merosin), and no adhesion occurred on bovine heart laminins and murine EHS tumor laminin. Further inactive substrates were fibronectin, nidogen, and collagen types IV and VI, indicating that the alpha 3A beta 1 integrin is a much less promiscuous receptor than thought before. By contrast, alpha 6A transfected cells adhered to all laminin isoforms when stimulated with TS2/16. Adhesion also occurred only on bovine kidney laminins in the absence of TS2/16. These results demonstrate that both alpha 3A beta 1 and alpha 6A beta 1 integrins are typical laminin receptors but that their affinity and activation dependence for binding to various laminin isoforms differ considerably.

Formation of hemidesmosomes in cells of a transformed murine mammary tumor cell line and mechanisms involved in adherence of these cells to laminin and kalinin.

Keratinocytes attach to an underlying basement membrane by adhesion junctions called hemidesmosomes. We have characterized a cell line, RAC-11P/SD, established from a murine mammary tumor, which differentiates into squamous epithelium and forms well defined hemidesmosomes. These hemidesmosomes contain the integrin alpha 6 beta 4 as well as the hemidesmosomal plaque proteins BP230 and HD1 and are associated with a matrix containing kalinin and laminin. We examined how these cells adhere to laminin and to kalinin present in matrices as well as immunopurified kalinin. We show that adhesion to laminin is energy dependent but does not require an intact actin-containing cytoskeleton. The affinity for kalinin proved to be greater and binding to kalinin was still observed when cells had been treated with deoxyglucose and azide to inhibit metabolic energy. Binding to laminin (or fragment E8), but not to kalinin was partially blocked by a monoclonal antibody specific for the integrin alpha 6 subunit, and only in the initial phase of adhesion. The antibody efficiently blocked adhesion to laminin of cells treated with the microfilament disrupting drug cytochalasin B, but only partially blocked the adhesion of cytochalasin B-treated cells to kalinin, while adherence of cells treated with deoxyglucose and azide to kalinin was blocked completely. The integrin alpha 6 beta 4 is redistributed to the basal surface during adhesion and then is organized into ring-like structures when cells are bound to laminin and localized into hemidesmosomes in cells adhered to kalinin. We suggest that anti-alpha 6 hinders the binding of the alpha 6 beta 4 integrins to its ligands laminin and kalinin, but cannot prevent adhesion when clustering of the integrin has become complete. In addition, there is evidence that adhesion to kalinin is mediated by a second receptor, which associates with the actin-containing cytoskeleton. The presence of such a second receptor is suggested because the cells can spread on kalinin, but not when they have been treated with cytochalasin B. On laminin spreading does not occur, irrespective of whether cells have been treated with cytochalasin B or not. The integrin alpha 3 beta 1, which has been identified as a receptor for kalinin but not for laminin, is strongly expressed in RAC-11P/SD cells and it seems likely that this integrin is responsible for spreading of cells on kalinin.