PI-3-kinase is an essential anti-apoptotic effector in the proliferative response of primary human epithelial cells to mutant RAS.

In contrast to its growth-inhibitory effect on primary mesenchymal cells, RAS oncogene activation induces a proliferative phenotype in normal human thyroid epithelial cells in vitro, consistent with its putative role in tumour initiation. Using this model, we previously showed that activation of the MAP kinase (MAPK) pathway is necessary, but not sufficient for the proliferative response to mutant (V12) H-RAS. Here we extend this work to show that another major RAS effector-- phosphatidylinositol-3-kinase (PI-3-K)--while also insufficient alone, is able to synergize with MAPK activation to mimic the effect of mutant RAS, albeit at reduced efficiency. Furthermore we show that PI-3-K is an absolute requirement for the proliferative response to RAS in these cells, acting via suppression of RAS-induced apoptosis. These data extend our understanding of RAS signalling in a clinically-relevant cell context and point to the use of PI-3-K inhibitors as potential therapeutic agents for targetting human cancers induced by RAS mutation.

RAS oncogene activation induces proliferation in normal human thyroid epithelial cells without loss of differentiation.

Neoplastic transformation of rodent thyroid epithelial cell lines by mutant RAS genes has been widely studied as an experimental model of oncogene-induced loss of tissue-specific differentiation. However, separate evidence strongly implicates RAS mutation as an early event in human thyroid tumour development at a stage prior to loss of differentiation. To resolve this controversy we examined the short- and long-term responses of normal human thyroid epithelial cells to mutant RAS introduced by micro-injection and retroviral transduction respectively. In both cases, expression of RAS at a level sufficient to induce rapid proliferation did not lead to loss of differentiation as shown by expression of cytokeratin 18, E-cadherin, thyroglobulin, TTF-1 and Pax-8 proteins. Indeed, RAS was able to prevent, and to reverse, the loss of thyroglobulin expression which occurs normally in TSH-deficient culture medium. These responses were partially mimicked by activation of RAF, a major RAS effector, indicating involvement of the MAP Kinase signal pathway. The striking contrast between the effect of mutant RAS on differentiation in primary human, compared to immortalized rodent, epithelial cultures is most likely explained by the influence of additional co-operating abnormalities in the latter, and highlights the need for caution in extrapolating from cell line data.

Activation of mitogen-activated protein kinase is necessary but not sufficient for proliferation of human thyroid epithelial cells induced by mutant Ras.

Given the high frequency of ras oncogene activation in several common human cancers, its signal pathways are an important target for novel therapy. For practical reasons, however, these have been studied mainly in the context of transformation of established fibroblast cell lines, whereas ras acts at an earlier stage in human tumorigenesis and predominantly on epithelial cells. Here we have developed a more directly relevant model - human primary thyroid epithelial cells - which are a major target of naturally-occurring Ras mutation, and in which expression of mutant Ras in culture induces clonal expansion without morphological transformation, closely reproducing the phenotype of the corresponding tumour in vivo. Transient or stable expression of mutant H-ras (by scrapeloading or retroviral infection) at levels which stimulated proliferation induced sustained activation and translocation of MAP kinase (MAPK) in these cells. Inhibition of the MAPK pathway at the level of MAPKK, by expression of a dominant-negative mutant or by the pharmacological inhibitor PD98059, efficiently blocked the proliferative response. Conversely, selective activation of MAPK by a constitutively-active MAPKK1 mutant failed to mimic the action of Ras and, although this was achievable with activated Raf, micro-injection of anti-ras antibodies showed that this still required endogenous wild-type Ras function. In contrast to recent results obtained with a rodent thyroid cell line (WRT), therefore, activation of the MAPK pathway is necessary, but not sufficient, for the proliferogenic action of mutant Ras on primary human thyroid cells. These data emphasize the unreliability of extrapolation from cell lines and establish the feasibility of using a more representative human epithelial model for Ras signalling studies.

Contrasting effects of activating mutations of GalphaS and the thyrotropin receptor on proliferation and differentiation of thyroid follicular cells.

The cyclic AMP pathway is a major regulator of thyrocyte function and proliferation and, predictably, its inappropriate activation is associated with a sub-set of human thyroid tumours. Activating mutations are, however, more common in the thyrotropin receptor (TSHR) than in its downstream transducer, Galphas. To investigate whether this reflects an inherent difference in their oncogenic potency, we compared the effects of retrovirally-transduced mutant (A623I) TSHR or (Q227L) Galphas (GSP), using the rat thyroid cell line FRTL5 and primary human thyrocytes. In FRTL5, expression of GSP or mutant (m) TSHR induced a 2 - 3-fold increase in basal levels of cAMP. This was associated with TSH-independent proliferation (assessed by both cell number and DNA synthesis) and function (as shown by increased expression of thyroglobulin (Tg) and the sodium/iodide symporter). In primary cultures, expression of mTSHR, but not GSP, consistently induced formation of colonies with epithelial morphology and thyroglobulin expression, capable of 10 - 15 population doublings (PD) compared to less than three in controls. Thus, while mTSHR and GSP exert similar effects in FRTL5, use of primary cultures reveals a major difference in their ability to induce sustained proliferation in normal human thyrocytes, and provides the first direct evidence that mTSHR is sufficient to initiate thyroid tumorigenesis.

Multiple connexin expression in peripheral nerve, Schwann cells, and Schwannoma cells.

Myelinating Schwann cells express the gap junction protein, connexin (Cx)32, which is present at the nodes of Ranvier and Schmidt-Lantermann incisures (Bergoffen et al. [1993] Science (Wash. ) 262:2039-2042). Following peripheral nerve injury, other members of the connexin gene family are also expressed (Chandross et al. [1996a] Mol. Cell. Neurosci. 7:501-518). This study surveys the connexin(s) expressed by rat sciatic nerve, cultured Schwann cells, and a mouse Schwannoma (TR6 Bc1) cell line. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification revealed a constitutive expression of mRNA encoding Cx32 and 43 but not Cx26, 37, 40, 45, and 46 in sciatic nerve. Mitogenic stimulation of cultured Schwann cells expressing Cx32 also resulted in the appearance of Cx43 mRNA. Schwannoma cells expressed exclusively Cx43 mRNA. These results were confirmed by Northern blot analysis. Functional gap junctions in cultured Schwann and Schwannoma cells were shown by analysis of the intercellular transfer of Lucifer yellow, although the coupling between primary Schwann cells was weak or undetectable. Treatment of primary Schwann cells with mitogens resulted in extensive dye coupling. An immunohistochemical study of adult sciatic nerve sections demonstrated Cx32 immunoreactivity at the nodes of Ranvier and in Schwann cell bodies. Lower intensity staining of Cx43 along the myelin sheath and Schwann cell bodies was also observed. Indirect immunofluorescent studies of Schwann cells treated with mitogens showed characteristic punctate cell surface staining of Cx43; Cx32 staining was detected mainly intracellularly. These results lead to the conclusion that in addition to the expression of Cx32 by normal adult sciatic nerve, low amounts of Cx43 protein are also present. The implications of the expression of two connexins by Schwann cells in Charcot-Marie-Tooth X-linked disease, a demyelinating peripheral neuropathy, are discussed.

Control of replicative life span in human cells: barriers to clonal expansion intermediate between M1 senescence and M2 crisis.

The accumulation of genetic abnormalities in a developing tumor is driven, at least in part, by the need to overcome inherent restraints on the replicative life span of human cells, two of which-senescence (M1) and crisis (M2)-have been well characterized. Here we describe additional barriers to clonal expansion (Mint) intermediate between M1 and M2, revealed by abrogation of tumor-suppressor gene (TSG) pathways by individual human papillomavirus type 16 (HPV16) proteins. In human fibroblasts, abrogation of p53 function by HPVE6 allowed escape from M1, followed up to 20 population doublings (PD) later by a second viable proliferation arrest state, MintE6, closely resembling M1. This occurred despite abrogation of p21(WAF1) induction but was associated with and potentially mediated by a further approximately 3-fold increase in p16(INK4a) expression compared to its level at M1. Expression of HPVE7, which targets pRb (and p21(WAF1)), also permitted clonal expansion, but this was limited predominantly by increasing cell death, resulting in a MintE7 phenotype similar to M2 but occurring after fewer PD. This was associated with, and at least partly due to, an increase in nuclear p53 content and activity, not seen in younger cells expressing E7. In a different cell type, thyroid epithelium, E7 also allowed clonal expansion terminating in a similar state to MintE7 in fibroblasts. In contrast, however, there was no evidence for a p53-regulated pathway; E6 was without effect, and the increases in p21(WAF1) expression at M1 and MintE7 were p53 independent. These data provide a model for clonal evolution by successive TSG inactivation and suggest that cell type diversity in life span regulation may determine the pattern of gene mutation in the corresponding tumors.

[Origin and progression of thyroid epithelial tumors: molecular and cellular mechanisms]. / Origine et progression des tumeurs épithéliales thyroïdiennes: mécanismes moléculaires et cellulaires.

Tumours of the thyroid follicular cell have been intensively studied as a model for investigating the molecular genetics of tumour development in epithelial cells. This review summarises the abnormalities of oncogenes and tumour suppressor genes which have been associated with the major sub-types of thyroid tumour. Although inevitably incomplete, the available data demonstrate well how successive genetic abnormalities drive clonal progression. Comparison of follicular and papillary tumours also reveals a fascinating correlation between genotype and pathological behaviour, consistent with a determining influence of the initiating oncogene on the "route" of tumour development. Finally, the emerging clinical implications are discussed.

Reinitiation of DNA synthesis and cell division in senescent human fibroblasts by microinjection of anti-p53 antibodies.

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the "young" morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.

An amphotropic retroviral vector expressing a mutant gsp oncogene: effects on human thyroid cells in vitro.

Point mutations of the gsp protooncogene (encoding the alpha-subunit of the Gs protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin-tagged Gln227-->Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA-tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10-20 well differentiated epithelial colonies/dish of 10(5) infected cells, with an average lifespan of 15-20 population doublings, only small groups of no more than 15-50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.

Calcium is transported into the lumen of pig thyroid follicles by fluid phase basolateral to apical transcytosis.

The lumen of thyroid follicles contains a high concentration of thyroglobulin, the thyroid prohormone and a high concentration of calcium (Ca2+). As thyroglobulin binds Ca2+, intraluminal Ca2+ is expected to be in free and protein-bound forms. In the present work, we have investigated the mechanism(s) by which Ca2+ could enter the lumen of thyroid follicles. 45Ca2+ uptake studies were carried out on reconstituted pig thyroid follicles (RTF) and pig thyroid cell monolayers (TCM) in primary culture, representing experimental systems with two compartments (cells + lumina) and one compartment, respectively. 45Ca2+ accumulation in RTF was rapid during the first hour of incubation and then slowly increased. Analysis of the uptake data with a "two compartments" model gave two kinetic constant values: k = 1.71 +/- 0.28 hr(-1) and k(-2) = 0.20 +/- 0.05 hr(-1) (n = 10). The slow uptake process accounted for 20-50% of the total RTF-associated Ca2+ after 24 hr. 45Ca2+ uptake by TCM was rapid and reached a stable level within 1-2 hr. Experimental data fitted with a "single compartment" model and gave a k(-1) value of 1.64 +/- 0.15 hr(-1) (n = 10) which was not statistically different from the k(-1) obtained for 45Ca2+ uptake by RTF. We then compared the kinetics of 45Ca2+ uptake by RTF with the kinetics of transport of fluid phase markers: [14C]-sucrose and Lucifer Yellow from the medium to the lumen of RTF. [14C]-sucrose and Lucifer Yellow uptakes by RTF appeared as slow processes compatible with the entry in a single compartment with k values of 0.32 +/- 0.06 hr(-1) (n = 3) and 0.23 +/- 0.015 hr(-1) (n = 3), respectively. These values were not significantly different from the k(-2) value obtained for 45Ca2+ uptake by RTF. These data suggest that thyroid follicles would possess two independent Ca2+ compartments: cells and lumen, and that the entry of Ca2+ into the lumen of follicles probably could take place by fluid phase basolateral to apical transcytosis.

Tolerance of high levels of wild-type p53 in transformed epithelial cells dependent on auto-regulation by mdm-2.

A significant proportion of human cancers express high levels of p53 protein in the absence of an underlying mutation in the gene. Using transformed (Vh1) and non-transformed (FRTL-5) rat thyroid epithelial cell lines as a model, we have examined the mechanisms by which high levels of wild-type p53 may be tolerated. Stable transfection with p53-dependent reporter constructs demonstrated that the 'excess' wild-type p53 in Vh1 cells is not associated with a comparable increase in p53-dependent transcription (though the response to u.v. irradiation is retained). Mdm-2, which binds p53 and inhibits its transactivation activity, is overexpressed in Vh1 cells in the absence of gene amplification and in a p53-dependent manner. Furthermore disruption of p53-mdm-2 complex formation in Vh1 cells by microinjection of an antibody to the p53-binding domain of mdm-2 resulted in a dramatic increase in p53-dependent transcription. Since only a small proportion of the p53 in Vh1 cells was found to be in complex with mdm-2 (the majority of unbound protein being in a latent form), this suggests that mdm-2 selectively binds a pool of p53 that would otherwise be active as a sequence-specific activator of transcription. We suggest that, in some types of tumour, the 'sensitivity' of the p53-driven mdm-2 feedback loop may be sufficient to prevent free, active p53 reaching the level required for growth arrest or apoptosis, making them an ideal target for therapies designed to disrupt p53-mdm-2 interactions.

Involvement of a membrane-bound form of glutamate dehydrogenase in the association of lysosomes to microtubules.

A 50-kDa membrane protein corresponding to a membrane-bound isoform of glutamate dehydrogenase was proposed as a molecular species that could mediate lysosome-microtubule interactions. This protein, isolated from purified lysosome membranes, is a peripheral membrane protein with an ATP-dependent microtubule binding activity. We have produced antibodies against the purified 50-kDa protein to investigate its role in the association of lysosomes to microtubules using a cell-free reconstitution assay and cell microinjection. Pretreatment of purified lysosomes with the antibodies inhibited the association of these vesicles to microtubules. The blocking effect of antibodies was demonstrated by a differential sedimentation method and negative staining electron microscopy, allowing us to quantify the amount of microtubules interacting with lysosomes and the proportion of lysosomes bound to microtubules, respectively. Affinity-purified antibodies microinjected into intact cells altered the distribution of lysosomes that appeared less clustered in the vicinity of nuclei. The antibody-induced lysosome dispersion was assessed by quantitative videomicroscope analyses. These data show that the 50-kDa membrane protein could act, through its microtubule binding activity, as a molecule of attachment of lysosomes to microtubules. This membrane-bound isoform of glutamate dehydrogenase could be involved in the microtubule-dependent perinuclear localization of lysosomes.

Evidence that transcriptional activation by p53 plays a direct role in the induction of cellular senescence.

Wild-type p53 is necessary for the growth arrest of human diploid fibroblasts (HDF) (and many other cell types) at the end of their proliferative lifespan. Although p53 may actively mediate senescence, possibly in response to telomere erosion, it is however equally possible that it is merely a permissive factor required for response to some other inducer. To address this question, we have generated stable transfectants of early passage HDF, represented here by clone LacZ21, in which expression of a beta-galactosidase reporter construct reflects p53 transactivation activity. During continuous passage, the proportion of beta-gal positive LacZ21 cells remained below 2% for 25 population doublings (pd), first became significantly increased after 29 pd, and thereafter increased rapidly, reaching a maximum of 88% in fully-senescent cells (32 pd), which exceeded the response observed following an optimum dose (20 J/m2) of u.v. radiation. Correspondingly, the proportion of cells incorporating bromodeoxyuridine (BrdU) (initially 45-50%) began to fall at 29 pd and thereafter dropped rapidly to below 1% by pd 32. There was therefore a near-perfect reciprocal relationship between reporter construct expression and DNA synthesis as cells approached senescence. Furthermore, a dominant-negative p53 mutant (introduced by retroviral transduction) rescued LacZ21 cells from senescence and generated colonies with extended lifespan in which beta-gal expression was totally abolished. These data, although not excluding the need for other p53 functions, strongly suggest that p53-mediated transactivation of growth regulatory genes is a direct trigger, rather than a permissive factor, for cellular senescence.

Endocytosis of albumin and thyroglobulin at the basolateral membrane of thyrocytes organized in follicles.

Serum proteins such as albumin are present inside thyroid follicles in both normal and pathological situations. To analyze the mechanism of entry of these proteins, we investigated the ability of polarized thyrocytes to internalize soluble molecules at their basolateral pole. Experiments were conducted on in vitro reconstituted thyroid follicles using BSA and pig thyroglobulin (Tg) coupled to gold particles for electron microscopy, conjugated to fluorescein for conventional and confocal fluorescence microscopy, or radioiodinated for biochemical measurements. Incubations were carried out at 37 C. BSA and Tg coupled to gold particles were rapidly internalized from the culture medium and sequentially found in small vesicles and early endosomes and in late endosomes and lysosomes. Fluorescence microscope analyses revealed that the majority of cells forming reconstituted thyroid follicles are capable of internalizing BSA and Tg, but that Tg was more efficiently endocytosed than BSA. Using radioiodinated ligands, it was observed that the endocytosis of Tg was 10 times higher than that of BSA. The internalization of [125I]Tg was inhibited by increasing concentrations of unlabeled Tg. In contrast, endocytosis of 125I-labeled BSA was independent of the unlabeled BSA concentration. Experiments performed at 4 C indicated the presence of a basolateral membrane binding activity for [125I]Tg; the Tg concentration that reduced the binding of labeled Tg by 50% ranged from 4-6 microM. These data are evidence of a process of internalization of soluble molecules at the basolateral pole of thyrocytes, with BSA being internalized by fluid phase endocytosis and Tg by selective endocytosis. Our findings explain how serum albumin can enter thyroid follicles and disclose a new cellular handling and transport pathway of Tg. We propose that selective uptake of Tg operating on molecules secreted at the basolateral surface of thyrocytes could control the amount of Tg released in the circulation.

Multinucleated cells can continuously generate mononucleated cells in the absence of mitosis: a study of cells of the avian osteoclast lineage.

The multinucleated bone-resorbing osteoclast has a hematopoietic origin. We have demonstrated previously that osteoclasts are derived from the monocytic lineage by fusion of mononuclear macrophage precursors. Using an in vitro-osteoclast differentiation model derived from pure populations of chick macrophage cultures, osteoclast-like multinucleated giant cells (MNGCs) can be formed by fusion following an active proliferation phase. However, after reaching a peak with 70% of the culture being MNGCs, a new round of expansion of the mononuclear cells is observed. The following experiments suggest that these mononuclear cells were derived directly from the MNGCs by a budding process, selectively from the central zone of the apical surface. After microinjection of the membrane-impermeable probe, Lucifer Yellow, into single MNGCs, initially only diffuse fluorescence, limited to the whole MNGC injected, was observed. However, after 24-48 hours fluorescent mononuclear cells were observed adjacent but distinct from the injected MNGC. To confirm that these mononuclear cells were indeed derived from a parent MNGC, single MNGCs were cloned into single wells. Within a week, the MNGC was surrounded by mononuclear cells, which eventually populated the entire well. These mononuclear cells could then give rise to a second generation of MNGCs following a three-week period of culture. To determine whether this process required mitosis, MNGCs were cultured for three days in the presence of the mitotic inhibitor, Ara-C, prior to microinjection with Lucifer Yellow. Fluorescent mononuclear cells were still seen to arise from a single injected MNGC under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein endocytosis by a kidney tubule suspension: metabolic requirements.

Endocytosis in the renal tubular cell is a permanent process serving the role of saving nitrogen from plasma peptides that are continuously cleared away by kidney glomerulus. Since small proteins appear in urine after strenuous exercise, it was hypothesized that renal ischemia impairs the tubular endocytic reabsorption of proteins. The aim of this paper is to describe a simple in vitro model of renal endocytosis and to use it in studies of endocytic metabolic requirements. The results show that rabbit renal proximal tubules in suspension are able to take up 125I-lysozyme, as well as RITC-lactalbumin. The fluorescent protein was taken up only by the ends of the everted tubule fragments, and accumulated into intracellular vesicles, demonstrating the luminal pathway of endocytosis. The amount of 125I-lysozyme taken up was equivalent to that taken up by isolated perfused tubules (Nielsen et al. (1986) Am. J. Physiol. 251, F822-F830). Anoxia decreased 12-fold the intracellular accumulation of 125I-lysozyme; however, the time-course of inhibition shows that only the late steps of endocytic accumulation are energy-dependent. Substrate deprivation studies suggest a specific role of glucose to sustain endocytosis. Lastly, renal uptake of 125I-lysozyme was shown to be strongly depressed by chloroquine, an alkalinizing agent of endosomes and lysosomes. We conclude that (1) renal tubules in suspension are a satisfactory model for endocytic studies in kidney; (2) suppressing oxygen and substrate supplies to kidney impairs endocytic tubular reabsorption of proteins.

Dynamic analysis of drug action on in vitro reconstituted thyroid follicle by microinjection of tracer molecules and videomicroscopy.

Thyroid cells isolated from the gland by trypsinization are capable in culture of reconstituting histiotypic structures, the thyroid follicles. This morphological differentiation requires the presence of the main thyroid regulator; thyrotropin. We have analyzed some structural and functional aspects of in vitro reconstituted thyroid follicles (RTF) using microinjection of fluorescent probes and videomicroscopy. This experimental approach allowed to visualize biological processes and actions of drugs, signalling factors, etc. in living cells. We describe here some examples of what can be studied with this powerful still-undervalued method. Microinjection of a cell-impermeant fluorescent probe of either high or low molecular mass into the lumen of RTF allowed to check the tightness of this compartment and therefore to analyze the control of tight junctions assembly. A small cell-impermeant probe like Lucifer Yellow microinjected into a cell was used to demonstrate and then to study the regulation of cell to cell communication via gap junctions. The presence of calcium in the lumen of RTF was detected by microinjection of a properly designed probe: Calcium Green which becomes fluorescent in the presence of the ligand. The lumen to cell transport or endocytosis of thyroglobulin, the thyroid prohormone, which is stored into the lumen of the follicles, is currently studied by microinjection of TRITC-labeled thyroglobulin. Coupled to image processing and videorecorder systems, kinetic analysis and quantitative measurements can be performed.