Patent Application: US-10939402-A

Abstract:
the method for repetitively determining a profile of quantitative features for different cells in a sample by collecting and analyzing information on the mass distribution in cells or portions of cells and comparing the collected information to values from a relational database is disclosed .

Description:
the present invention is an improvement on previous work by the inventor herein which led her to discover that transformation - dependent changes in cell shape are caused , in part , by a perturbation of vesicle traffic ( 1 ). a comparison of normal and oncogenically transformed cells indicated that the latter had an excess of membrane - bound vesicles trapped in the peripheral cytoplasm . for the sake of brevity , these native differences are referred to as the signature - type of transformed cell . the signature - type could be modeled by exposing cells to one of four different classes of agents . one class was represented by a phorbol ester tumor promoter , which was thought to affect cells by activating protein kinase c ( pkc ). an additional class of compounds affected mts . the remaining two classes of agents affected ion and proton transport and caused alkalinization of intracellular organelles . work from other laboratories showed that cells treated with bioactive phorbol esters accumulated contents in the endocytic pathway to an unusual extent ( 2 ). since the other agents also interrupted normal endosome processing , this phenotype appeared common to cells treated with all four types of agents ( 1 ). the earlier research had been done by culturing cells on interferometers and digitizing their interference contours . from the shapes of three such contours , values for a total of 102 variables were extracted , of which only a small subset was used in classifying the treated samples . these variables provided a means of categorizing each cell according to its shape characteristics , so that it could be known which agents mimicked the effects of cancerous transformation . until the present invention , however , it was not possible to break down such transformations further into their component changes using the original classification protocol . this was a formidable obstacle to gaining a full understanding of the physiological defects of transformed cells . in order to learn what defects were enhanced by each of the above agents , to cause mimicry of the transformed phenotype , it was essential to make qualitative as well as quantitative distinctions among cell types . it was also an essential condition to determining whether aberrant membrane processing was the major change that occurred when cells adopted the signature - type . improvements in classification methodology are used to group like changes among the variables together and represent them by a single factor . when the original contour shape data are broken down by factors , we noted that some of the latter data corresponded to observable morphological features of cells ( 3 ). fig5 - table 1 defines features described by the various factors used in classification exercises . notable among the factors was # 4 , which reflected the prevalence of filopodia or microspikes , cell specializations that are induced in other model systems by activation of the g protein , cdc42 ( 4 - 6 ). thus , factor # 4 values increased with the increasing frequency or length of these actin - based structures . it is possible that other factors reflected the retention of extracellular fluids in endosomal compartments . possible candidates for such a role , for example , were those indexing the prevalence of hollowed - out regions (# 13 ) and the variance in projection size (# 11 ), respectively , in the second contour . factor - based analysis has the potential of allowing the effects reported earlier to be broken down and to distinguish subtypes among the different treatment groups , by the simple expedient of comparing factor values for treated samples and controls . it can also be determined whether there is an exact correspondence between the changes caused by agents simulating the signature - type and the bona fide transformed cell . the results show that two classes of agents , ionophores and mt inhibitors , cause a reduction in values of factor # 4 . the factors that might have been expected to change , however , were those related to the second contour (# 11 an # 13 ) or to bumpiness in both the uppermost contours (# 2 ). the results of these experiments were intriguing in that they showed an underlying coordination between physiological processes and otherwise unrelated morphological features . there are several ways of making disproportionate changes in factor # 4 . one of them requires compensatory changes of membrane trapping in early endosomal compartments . other compounds causing mimicry of the signature - type had more subtle effects , which did not rely on alterations in one or two specific factors . of the various pharmaceutical agents that were found to model the signature - type , all showed a promoter - like effect ( reviewed in ref . 1 ). however , there was preliminary data suggest that reversal of the transformed phenotype occurred in cells treated with an mt inhibitor combination ( ic ) consisting of colchicine with a molar excess of paclitaxel ( heckman c a and plummer h k iii : anticancer res 12 : 1915 - 1916 , 1992 ). at the time this result was obtained , little could be done to elucidate the features that were changed to create this phenotype . however , as shown by the present invention , the ic - mediated reversal is real and appears to rely upon reversal of the same effects as are promoted by treatment with pma or weak bases . moreover , the effects were specific to one cell line , and they were opposite in direction to the effects of paclitaxel itself on sharp , tapering features of the cell edge . the results implied that early endosomal processing occurs normally during ic - mediated reversal of the signature - type . additional data showing mt localization were gathered by immunofluorescence techniques . these show that the sharp , tapering morphogenetic features which are notably reduced in ionophore - and colchicine - treated cells relate to mt positioning at the cell edge . earlier experimental results suggested that all four classes of pharmacological agents that affected the transformed phenotype interfered with endocytic vesicle processing ( 1 ). in order to see whether any of these treatments yielded an exact replica of the signature - type , it was essential to be able to compare the morphological features represented in two samples on a quantitative basis . this was made possible by a method of phenotype classification which , although based on the same contour shape variables as formerly used , enabled us to break down each cells &# 39 ; phenotype into new variables called latent factors ( 3 ). with the new method of the present invention , notable discrepancies between the type created by experimental treatment and the signature - type of bona fide transformed cells were found . when the morphometric patterns of the cells in experimental samples were subjected to analysis based on factor values , the outcome suggested that it was possible to interpret the four classes as affecting a few key activities . one of these was composed solely of factor # 4 . others reflected additional cell edge features (# 7 ), the arrangement of organelles in the interior of the cell (# 2 , # 11 , # 13 ), or cell spreading (# 12 , # 16 ). since these data might provide clues as to the regulatory pathways affected by the transformation event and how they contribute to deregulated growth , a detailed analysis of the four classes of agents and their effects on physiological processes was performed . both dissociation of ligand - receptor complexes and ligand transfer to the lysosome can be inhibited by monensin and weak bases ( 19 , 20 ). likewise , mt depolymerization interfered with the separation of ligand - receptor complexes and inhibited the transfer of proteins into late endosomes and their degradation in the lysosome ( 21 , 22 ). since both monensin and colchicine reduced the prevalence of sharp projections in the shape phenotype , an interference with ligand - receptor processing was linked to this particular feature of the transformed cell . although this effect was first reported in the current research , it is consistent with previous results showing that monensin treatment mainly affected variables of the cell edge whereas the signature - type involved changes in both of the lowermost contours ( 1 ). since the data suggested that enhanced na + or k + pump activity and the consequent increase in k + concentration were not responsible for cells taking on the signature - type , the outcome of the ionophore experiments appeared to rely on the piling - up of contents in endosomes . while monensin , a na + / h + / k + ionophore that carries monovalent cations across membranes in exchange for protons , causes the trapping of membrane receptors in endosomes , there is also a failure to recycle to the cell surface ( reviewed in ref . 1 ). this trend is duplicated by the membrane as a whole , as indicated by studies from another laboratory demonstrating inhibition of fluid - phase marker uptake ( 23 ). these data suggested that a fraction of the plasma membrane became immobilized in the trapped form . it is confirmed herein that there was a reduction in the rate of uptake in the longer term . despite the fact that the uptake of the marker , horseradish peroxidase , by iar20 pc1 cells was not inhibited immediately after monensin treatment , it was inhibited within 30 min after initial exposure . however , marker was accumulated in larger compartments in monensin - treated iar20 pc1 cells than in control cells ( data not shown ). therefore , endocytic rates were reduced as a secondary effect of membrane trapping . moreover , traffic in a newly synthesized pool of membrane that is ordinarily available for insertion into the plasma membrane may be impaired by a blockade of endocytosis - linked exocytosis . a mechanism that couples transport in the inward and outward pathways of mt - mediated vesicle traffic may prevent traffic in the constitutive secretory pathway ( 24 , 25 ). two additional ionophores that have a monensin - like effect on sharp projections of the cell edge were found . one of them , gramicidin , was a selective transporter of sodium and was expected to equilibrate the na + concentration . the second , valinomycin , is selective for k + over na + and is used to short - circuit the membrane potential ( 26 ). the fact that all three ionophores , monensin , gramicidin , and valinomycin , elicited similar cell phenotypes suggest that all of the ionophores act by elevating the ph of an intracellular compartment . thus , in all three cases , trafficking was thought to be affected through the flux of monovalent cations into organelles . as indicated by the example discussed herein , colchicine resembles these ionophores in its qualitative effect on the factor - based phenotype . however , the mechanism employed in this case no doubt differed from that utilized by ionophores . mts are used . by cells to direct the traffic of both secretory and endocytic transport vesicles , and so their depolymerization is thought to inhibit a later step of endosome processing , namely that which mediates the separation of vesicles containing ligand only from those having ligand - receptor complexes ( 27 ). as was the case for monensin , mt - depolymerizing agents did not inhibit the instantaneous uptake of fluid - phase markers , but in cells exposed for longer times , appeared to repress endocytosis ( 28 ). thus , the colchicine effect probably reflects the same membrane pooling process as was induced by monensin . the prevalence of sharp features in ionophoreand colchicine - treated cells is reduced whenever endosomal processing is terminated at an early stage . the defect in processing leads to a reduction of sharp , tapering projections . although the mechanism by which the sharp features are produced is unknown , two explanations are possible consistent with the current understanding of membrane processing . one is that receptors at the plasma membrane play a role in organizing the sharp projections . if , during net membrane internalization , these receptors become pooled internally by membrane trapping , fewer sharp projections could be maintained at the cell edge . the second possible explanation is based on a current concept of coupling between the endocytic and exocytic pathways . according to this hypothesis , the process of membrane trapping would lead to a secondary reduction in the amount of membrane secreted . a reversed coupling of trafficking has been demonstrated in studies of yeast , where blockage of the early secretory pathway causes membrane trapping in the endocytic pathway ( 29 ). if actin microspikes are formed through site - directed membrane insertion at places specified by exocytosis of golgi - derived vesicles , as is thought ( 30 ), then the features may be reduced owing to the failure of exocytosis to proceed . since new membrane would not be available to be added at the cell edge , the fine , tapering projections would shrink and # 4 values would decline . to evaluate the above data in relation to the dynamic process of filopodia formation , it is important to gain further information on treatments that altered factor # 4 values . like other mt inhibitors , paclitaxel slowed the transport of endocytosed fluid - phase marker to the lysosome in some model systems ( 31 ) and caused the trapping of ligand - receptor complexes ( 32 ). these results suggested a membrane pooling effect similar to those caused by colchicine and monensin . in addition , investigators studying primary hepatocytes found that paclitaxel promoted a reorganization of the apical actin filament network and the extension of filopodia ( 31 ). the 1000 w cells , which showed an increased prevalence of sharp , tapering features , resembled hepatocytes in this respect . nevertheless , the effects were not strong enough to change factor # 4 values . on the other hand , paclitaxel was effective at promoting the signature - type in liver cells if delivered over a prolonged period of time . when the variables contributing to factor # 4 were subjected to analysis , to explore the differences in the two cell lines , it was found that liver cells showed a shift in these variables like that observed in ionophore - or colchicine - treated cells . the same set of variables for 1000 w cells , however , changed in the opposite direction to that found in iar20 pc1 , indicating an increase in size or frequency of tapering projections . although it is possible that cells responded to paclitaxel with increased vesicular trafficking rather than membrane pooling , it was an unlikely explanation of the data . rather , the effect on filopodia formation may have involved a better integration of the pathways of endocytic and exocytic vesicle trafficking is 1000 w cells . alternatively , actin may have been mobilized by partial dissolution of stress fibers , adding onto sites at the cell periphery . rounding - up of the cell (# 12 ) and contractility are affected by mt inhibitors . an increase in the angle at which the cell rises from the substrate ( factor # 12 ) was among the morphometric features that characterized signature - type cells . the values of # 12 were disproportionately affected by mt inhibitors , although not by ionophores . the two mt inhibitors &# 39 ; effects on this factor were also opposite in direction , since colchicine mimicked and paclitaxel counteracted the change seen in the original signature - type . studies from other laboratories reveal the shift in # 12 as being a probable result of rho - mediated changes in actin architecture . rho activation takes place through a cascade of events . the first step was activation of rho by mt depolymerization and the second , that of rho kinase by rho . finally , rho kinase is thought to mediate the inhibition of myosin phosphatase , and thereby allow phosphorylated forms of myosin light chain kinase and / or myosin regulatory light chain to accumulate ( 33 - 35 ), causing components to be added to actin stress fibers and focal adhesion sites ( 36 , 37 ). the mt - bound rho guanine nucleotide exchange factor , gef - h1 , which is thought to be activated upon mt - depolymerization , is a candidate initiator for these mt - dependent effects ( 38 ). the effects of paclitaxel were opposite to those of the depolymerizing agents in other respects as well . in cultured ovarian granulosa cells , paclitaxel itself impaired the formation of stress fibers and actin was mobilized instead to the cell periphery ( 16 ). a comparable effect is found with cdc42hs overexpression , which causes a reduction in stress fibers along with an increase in diffuse actin ( 5 ). such effects are attributed to the possibility that actin mobilized when stress fibers are dissolved , can contribute to features with other actin - based architectures . in an ic , paclitaxel prevented the elevation of actin stress fibers and focal adhesion sites that had otherwise been found after treatment with the depolymerizing agent alone ( 37 , 39 ). thus , the results showed that an mt - bound molecule was i ) responsible for rho activation and ii ) released from mts by colchicine — a release that was prevented if paclitaxel was also present . stress fibers are elaborated in other model cell lines after colchicine treatment , and indeed , this agent promoted greater quantitative shifts in the signature - type than the taxanes did in the examples herein . this was consistent with the known changes in factor # 12 and suggested that the state of contraction associated with elaboration of stress fibers , was associated with increasing values of # 12 . on the contrary , the mechanism of paclitaxel - mediated factor # 12 reversal is assumed to rely , in part , upon its inhibiting or preventing the release of the postulated rho activator . the fact that taxanes elevated surface smoothing phenotype in one cell line and not the other is difficult to explain . it may depend on diverse causes of coupling between sharp features and membrane trapping and whether this effect may be outweighed by stress fiber actin mobilization on occasion . the difference may relate to the preprogrammed state of differentiation of the cultured cells . pma can alter phenotype without disproportionate effects on any single factor &# 39 ; s value . the effects of all three classes of agents discussed above appeared to be all - or - nothing in nature , i . e ., each agent either promoted the signature - type or was ineffectual . pma - stimulated cells showed more complex changes since , in this case , different factors were disproportionately affected at different times but returned to their original values after a prolonged course of pma exposure . the time course of changes complemented the cycle of ruffling stimulation and suppression . ruffling stimulation by phorbol esters has been observed by a number of workers using a variety of other cell types ( 40 - 42 ). in 1000 w tracheal cells , pma - mediated ruffling suppression began by 10 h after the initial exposure and became severe by 15 h pma exposure ( 1 ). compared to its direction of change in transformation , factor # 4 values underwent reversal at the 10 - h interval and therefore , increased as ruffling frequency declined . a similar cycle of ruffling stimulation occurred after growth factor administration , but this activity was found to be downregulated within an hour or less ( 43 ). depressed values of another cell edge feature , # 16 , appeared to reflect a decline in lamellar cytoplasm , as the cell edges retracted at the 5 - h treatment interval . since phorbol esters act as surrogates for diacylglycerol , which is in turn the endogenous activator of pkc , the latter is implicated in the mimicry of transformed - type features . diacylglycerol is ordinarily released when growth factor receptors stimulate the enzyme , phospholipase c , to hydrolyze its substrate , an inositol phospholipid . by activating pkc directly , the bioactive phorbol esters circumvent these initial steps of signal initiation . the subsequent steps in the signaling process are mediated by pkc substrates or by binding of the regulatory sequence of pkc to other proteins ( reviewed in ref . 44 ). included among the latter is pld , an enzyme that recruits and binds activated pkc to specific sites on membranes . pkc activates this enzyme synergistically with small g proteins , rhoa and arf , which bind pld directly ( 45 - 47 ). since pkc activation enhances the internalization of certain cell surface receptors ( 48 ), as well as markers for the extracellular medium ( 49 , 50 ), the data suggest that pld accelerates membrane processing in endocytic vesicles following their release from the plasma membrane . alterations in trafficking may be one of the ways in which pma causes receptor downregulation and reduces the number of receptors at the cell surface ( see below ). pma - induced receptor processing , however , seems to differ in some respects from processing of ligand - receptor complexes during regular signaling ( 51 , 52 ). this effect may relate to the fact that ligand - bound receptors with a short residence time in the endocytic pathway escape being routed to the lysosome ( 53 , 54 ). pma profoundly alters the kinetics of both membrane uptake and processing . while pma caused contents to pile up in the endosomal compartment because of a stimulation of fluid - phase uptake , it is still unclear whether there is any change in subsequent trafficking . other workers have shown that receptor internalization is more sustained in phorbol ester - treated cells than in untreated cells ( 55 ). in such cells , some receptors , for example , cd4 , were preferentially routed to a lysosomal compartment along with the fluid - phase contents of vesicles . in lymphocytes and macrophages , both these constituents appeared to follow a pkc - mediated default pathway ( 56 , 57 ). on the contrary , the t - cell receptor escaped degradation after pma - induced receptor downregulation , although it was normally degraded when bound to its ligand ( 52 ). indeed , some ligand - bound receptors are routinely internalized into a different compartment from that containing rapidly recycling endosomal components ( 58 ). thus , one distinction that remains to be made in trafficking studies is whether the receptors selectively down - regulated after pma are accommodated in a specific endocytic compartment . the exact effects of pma on receptor processing may be related to the known translocation of the major phosphorylated protein , myristoylated alanine - rich c kinase substrate , from the plasma membrane to the lysosome in treated cells ( 59 ). the routing mechanism following pkc activation needs to be clarified in different cell types in order to relate it to mechanisms of cell shape change . although the changes in factor values stimulated by pma are complex , they are related to the physiological defects that characterize transformed cells . although most of the factor - based alterations in cell phenotype were reversed again at 10 - or 15 - h times , it was found that # 2 adopted the signature - type value by the 10 - h interval . further , this particular factor reflects the well - known downregulation of receptor content which is known to occur in some cells . at other time intervals , statistically significant changes in signature - type occurred which were not accompanied by a change in the value of any factor used for classification , and the properties of these samples closely resembled those of the bona fide transformed cell . the mechanism by which weak bases affected the signature - type was instructive in relationship to that of ionophores that rendered the endocytic compartments permeable to ions . comparing these agents , it was found herein that the former caused a well - balanced mimicry of the transformed cell . both agents were postulated to elevate the ph of intracellular compartment and thus retard the loading of endocytic vesicles into mts . nevertheless , the effects of weak bases appears incomplete , since work from other laboratories suggests that a substantial fraction of the endocytic vesicles formed undergo retrograde transport ( 19 , 61 , 62 ). polarized cells , in the presence of chloroquine , show more vesicle traffic than usual from the basolateral to the apical surface suggesting that the treatment also may interfere with the routing of vesicles to the lysosome ( 63 ). the known effects of weak bases indicated that endocytic vesicle trafficking continued to some extent , and in this respect , the treated cells would bear a resemblance to those treated with pma . effects of simultaneous exposure to two mt inhibitors . in addition to pma and weak bases , a combination of mt inhibitors could cause a change in signature - type without there being disproportionates changes in any factor employed in the equation for classification . the effects of the paclitaxel and colchicine combination were specific to the cell line studied , and the signature - type shift only occurred in iar20 pc1 liver cells . paclitaxel , a well - known chemotherapeutic agent , has diverse effects on mt physiology . it inhibits tubulin disassembly , enhances mt stability , and inhibits the binding of mt accessory proteins ( 64 , 65 ). in intact cells , it also inhibited the tyrosinolation of β subunit of tubulin ( 13 ). treatment of iar20 pc1 cells with taxanes mainly affected the combination of factors # 1 and # 7 , but these effects are not well understood . paclitaxel &# 39 ; s ability to decrease the frequency of vesicle transport on mts ( 66 ) no doubt accounted for some of its effects on the factor values . however , having fewer vesicles being transported would be expected to enhance the roughness of boundary details , especially in the second contour , whereas the changes suggested a response like that of transformed cells where surface smoothness increased . except for a factor measuring hollowed - out regions in the second contour (# 13 ) in cells treated with the ic containing cephalomannine , such smoothing effects were lacking in the ic - treated cells . whereas exposure to a number of different agents caused cells to replicate or model the mass distribution of the signature - type , the combination of paclitaxel and colchicine was the only treatment that caused type reversal . it is likely that values of several factors have been changed in order to achieve a difference between experimental and control samples , but that such differences were too small to be statistically detectable . results of treating with cephalomannine , instead of paclitaxel , were used to compare the features that were affected differently by the two taxanes . variables indicating smoothing of projections on the first and second contours were affected when ics were made with taxanes other than paclitaxel . the results suggested a broadening effect on the microspike structures , although less marked than the effects induced by colchicine and ionophores . although ic of paclitaxel and colchicine had no statistically significant effect on the values of factor # 4 , a study of the primary variables that contributed to factor # 1 showed that some of them were changed . thus , if the mts functionally integrate with stable actin structures , as is commonly thought , it can be assumed that this arrangement may be affected during reversal . other laboratories reported that paclitaxel , whether delivered alone or in an ic with a depolymerizing agent , caused rearrangement of the mt array . as long as paclitaxel was in molar excess over the depolymerizing agent , it suppressed the organizing ability of the mt - organizing center ( 67 , 68 ). observations of ic - treated cells used in this research confirmed that the patterns of mt redistribution differed remarkably f rom those induced by paclitaxel alone . these patterns were well correlated with the reversal effect , since mts radiated from local centers in the cytoplasm only in cells that were exposed to the ic made up with paclitaxel . growth factor - induced changes in signature - type . while exposure of cells to pma short - circuited some of the initial steps of signaling , it could induce changes in receptor processing and prolong the residence time of internalized vesicles in the cytoplasm . in order to determine how this abnormal form of signaling compared to steady - state signaling in cultured cells , we altered the ambient insulin concentration in some experiments . insulin was known to stimulate endocytic trafficking and fluid - phase endocytosis ( reviewed in ref . 69 ). this , however , had a minimal effect on phenotype . whereas the signature - type cells drifted upward with higher levels of hormone , the differences were not statistically significant . in order to determine whether signature - type expression could still be induced after the growth factor - driven pathway was maximized , cells were treated with colchicine . the difference between untreated and colchicine - treated cell phenotypes decreased , indicating that cells at high levels of supplementation were less responsive . the results implied that , if signaling were driven by higher and higher concentrations of insulin , a failure in the cells &# 39 ; responsiveness to colchicine would be seen . thus , it is believed that the elevation of signaling has a similar ability to activate rho as is typically induced by mt depolymerization . whether this effect was mediated by an increased availability of guanine nucleotide exchange factors remains to be determined . downstream of insulin signaling , a novel isoform of pkc was implicated in the stimulation of gene transcription ( 70 ). it is believed that long - term insulin supplementation up - regulates rho activity but that the effect was counteracted by another regulatory mechanism , possibility working at the transcriptional level . this would account for the fact that insulin supplementation failed to induce a significant change in the signature - type . the following examples are provided merely to further illustrate the present invention . the scope of the invention shall not be construed as merely consisting of the following examples . cell lines and interference preparations . equations were developed to define changes in the signature - type for two epithelial cell lines that underwent transformation over a prolonged time of in vivo culture . although a different equation was developed in each case , there was considerable overlap in the factors employed as variables ( 3 ). iar20 pc1 was a clonal derivative of a line called iar20 , which was cultured from liver cells of a bd - vi rat . the other line , 1000 w , was generated from the tracheal epithelium of a fisher 344 rat after topical treatment with 7 , 12 - dimethylbenz ( a ) anthracene . the lines become tumorigenic after 11 and 16 months of being maintained in culture , respectively ( 3 ). liver cells were grown in william &# 39 ; s e medium supplemented with 10 % fetal bovine serum , whereas the tracheal cells were grown in waymouth &# 39 ; s medium containing fetal bovine serum plus 0 . 1 μg / ml insulin and 0 . 1 μg / ml hydrocortisone . the cells were maintained and subcultured as described elsewhere ( 1 , 3 ). for experiments with insulin supplementation , insulin concentrations were raised to 1 and 10 μg / ml and maintained for two passages before the cells were used . information about shape features was derived from interference images . these were generated by culturing cells on anodized tantalum substrates , and then imaging them in reflected light . cells were plated on substrates in replicate dishes , left overnight , and then exposed to experimental agents or to the solvent vehicle alone . after incubation with the agents for 2 - 5 h , samples were recovered , fixed in buffered 3 % glutaraldehyde ( ph 7 . 3 ), and air - dried . randomly selected cells were imaged in a zeiss universal microscope , and the outlines of their three lowermost interference contours were digitized . a database of 102 variables for contour shape was obtained for each cell , representing its mass distribution . each experimental treatment was represented by a total of 50 cells . classification procedures . the classification methods employed a subset of the contour shape variables . each 50 - cell sample was submitted to multiple linear regression analysis for classification alongside a group of cells representing normal and transformed reference samples . the latter were obtained from the lines that underwent transformation as described above . because the signature - type value was assigned relative to these samples , the solutions were in units of predicted time in culture . after obtaining such a value for each cell in an experiment , the differences in mean predicted times for treated and control cells were calculated . the sum of the squared standard deviations , divided by the number of observations , n , was used to compute the confidence limits of the mean : where s was the standard deviation for each sample . dividing the difference between the means by the square root of the above expression yields a value that can be related to the degree of confidence , 1 - α . for example , a z alpha / 2 exceeding 2 . 33 would indicate a large difference between the confidence limits of the mean for experimental and control samples . this value was equivalent to a probability that the two samples differed of p ≦ 0 . 02 . again , classification of samples from experiments employed multiple linear regression methods , based on factors scores rather than on contour shape variables . factors were generated by combining into a single database , the contour shape variables from highly malignant epithelial cells and precancerous lines that change progressively , eventually become neoplastic . the database contained values of 102 variables for 200 cells each from four cell lines , thus ensuring that values of the factors reflected a wide variety of shape phenotypes . principal components were extracted and transformed by a pattern of rotation to produce latent factors , which described variances that had thereby been rendered orthogonal ( 8 ). factor scores for each cell represented in the database to be estimated . these factor scores from known iar20 pc1 or 1000 w cells are used as a reference to classify data from short - term experiments on the respective samples . the factors used in classification were selected by the sas reg procedure ( 8 ). equations describing the relationship among factors were unique to each line ( 3 , 7 ). predicted time - in - culture values were obtained by submitting the experimental cells to analysis , as described above for simple variables . the statistical significance of differences among sample means within each experiment was determined by the duncan multiple range test . to determine whether any factor &# 39 ; s values differed among treatment groups within an experiment , the glm and model discriminant analysis procedures of sas were used . factor scores for dependency on the transformed state of each line by computing pearson &# 39 ; s product - moment correlation with time , using s - plus software ( statistical sciences , inc , seattle , wash .) were also tested . in further evaluating the factors used in the classification equations , a few of the factors were entered into the equations by multiple linear regression selection but were not well correlated with transformation . presumably , these were entered because they counterbalanced fluctuations in the value of one or more factors that were well correlated with transformation . examples of this effect include factor # 1 , which indexed massive protrusions or eroded features in the upper contours . these features became less prevalent in the signature - type of iar20 pc1 cell but showed no correlation with time in the 1000 w line . another example was the factor for variance in size of features in the second contour , # 11 , which was used in classifying cells from the tracheal line . as it defined features that were changed in the signature - type of 1000 w , along with factor # 2 , which described the number and size of projections and invaginations in the uppermost contours , it was thought to counterbalance fluctuations in the values of # 2 . a similar effect was noted with # 8 , a factor for sharp features in the second contour , which was included in the classification equation for iar20 pc1 cells , although it showed no significant correlation with transformation in this line . chemicals . quinidine sulfate , gramicidin d , valinomycin , monensin , ouabain , and colchicine were obtained from sigma - aldrich company ( st . louis , mo .) and used as previously described ( 1 ). cells were exposed to these agents at concentrations indicated in the text . all stock agents were dissolved in ethanol and stored at − 20 ° c . baccatin iii , cephalomannine , and paclitaxel were gifts from n . r . lomax and m . suffness , drug synthesis and chemistry branch and natural products branch , respectively , of the developmental therapeutics program , division of cancer treatment , national cancer institute . pma and 7 - deoxytaxol were obtained from lc laboratories ( woburn , mass .). cells were exposed to baccatin iii at a concentration of 9 μm , whereas the other taxanes were used at concentrations of 6 - 18 μm . no concentration - dependent differences were observed within this range . immunofluorescene localization of β - tubulin . iar20 pc1 cells were subcultured onto sterile glass cover slips as described above . replicate samples were exposed to test agents for 2 - 5 h alongside controls , and then fixed and left in methanol at − 70 ° c . until used for the immunofluorescence procedure . to visualize arrays of mts , cells were exposed to a mouse monoclonal anti - β tubulin antibody ( nycomed amersham , little chalfont , buckinghamshire , uk ), made up at a 1 : 300 dilution . we used a 1 : 100 dilution of p3 × 63 monoclonal antibody from p 3 myeloma ( generous gift from j . lessard ) as a non - specific control . the secondary antibody , eitc - conjugated goat anti - mouse immunoglobulin g ( us biochemical corp ., cleveland ), was used at a dilution of 1 : 2000 . cover slips were processed and then mounted on a slide in a solution of 1 , 4 - diazobicyclo [ 2 . 2 . 2 ] octane ( sigma ) made up to 25 mg / ml in glycerol . the samples were photographed on a zeiss axiophot microscope equipped with a 63x planapo lens . images were recorded on kodak tri - x or t - max film . all of the drugs , including paclitaxel , cephalomannine , colchicine , and baccatin iii , were studied to see if they affected the arrangement of mts . except for a tendency to develop more prominent bundles with time , no structural differences were noted in cells depending on the duration of exposure to the taxanes . mt arrangement was unaltered after treatment with the above - mentioned ionophores and transport inhibitors . immunofluorescent staining in cells treated with non - specific control immunoglobulin was negligible and failed to localize on any cytoskeletal structures . results : since some of the features changed in transformed cells were represented by discrete factors , each experimental treatment was broken down by factor values and it was determined whether each pattern of mimicry was attributable to a specific factor . using data collected from a progressive series of pma - exposure intervals on 1000 w cells , it was found that the signature - type was maximally expressed at 5 h and then underwent gradual reversal so that , by 10 h after treatment , the samples were classified as normal again . the results were similar to those reported earlier based on classification by values of single variables ( 9 ). in the factor - based classification , predicted times were 612 , 648 , 663 , 668 , and 604 days , respectively , for samples collected at times zero , 30 min , 2 h , 5 h , and 10 h after exposure . accumulation of fluid - phase contents was also enhanced over a similar time interval ( 1 ). to determine whether this time course involved changes in specific morphological components , the values of those factors that were relied upon most heavily to identify the signature - type were analyzed . factor # 1 was significantly elevated for a brief period after exposure , but reverted to control values within 2 h ( data not shown ). factor # 2 values were significantly reduced below control at the 10 - h time point . other strong predictors of the signature - type , including # 4 and # 12 , showed values that were indistinguishable from those of control samples at times between 0 . 5 and 5 h . in one experiment , some factors were altered over the control , but they were those that accounted for relatively little of the original change in signature features . both # 7 , an indicator of knob - shaped projections on the cell edge , and another weak predictor , # 16 , an index of the size variability in projections at the cell edge , adopted values like those of transformed cells . in a second experiment , none of the factors relied upon for classification showed a significant difference from the control values at the 5 - h time of phenotype expression ( table 2 ). ionopores and colchicines reduce the frequency of sharp projections . certain agents that alter the content of intracellular ions and protons cause fluid - phase contents to accumulate in cells by blocking traffic in the endocytic pathway . monensin is the agent that has been most widely used to study such effects . the phenotypes that were produced in cells by treatment with monensin , gramicidin , and valinomycin , were compared to determine whether they were qualitatively similar . monensin , which had been found to enhance the signature - type in early work , equilibrated both protons and monovalent ions across the membrane . gramicidin had equal affinities for na + and k + but resembled monensin in that it allowed intracellular levels of na + to rise ( 10 ). valinomycin predominantly bound and transported k + and was used to induce energy - dependent h + ejection from organelles ( 11 ). with factor - based analysis , both valinomycin and gramicidin d shifted the phenotype of iar20 pc1 liver cells to values of 336 and 387 days , respectively , compared to 283 days for control ( fig1 ). when the values of the individual factors used for classification were analyzed , the following differences were found . a high concentration of monensin used in previous studies ( 1 ) was found to cause substantial changes in several factors . a change in factor # 4 was common to these cells ( data not shown ), however , and to those treated with gramicidin and valinomycin ( fig6 - table ii ). the value of # 8 was also affected by valinomycin treatment in one experiment shown in table ii , as well as by exposures of 24 μm monensin ( data not shown ). gramicidin was unique among the ionophores in that it caused an increase in factor # 7 values , indicating a greater prevalence of knob - like structures at the cell edge . the above results show that the exact composition of the monovalent ion transported was irrelevant to signature - type induction . an additional anticipated outcome after monensin exposure , at least , was membrane hyperpolarization which could be expected to result from stimulation of plasma membrane na + , k + pump activity . the possibility that this was instrumental in signature - type mimicry was ruled out by administering monensin along with ouabain . the latter blocked monensin - induced acceleration of 86 rb + entry in other model systems ( 12 ) and thus prevented hyperpolarization . when cells were exposed to 24 μm monensin alone and classified on the basis of factor values , their average predicted time was solved as 344 days , compared to 318 days for the control . administration of monensin together with 50 μm ouabain caused the phenotype to shift to 347 days , a value that differed significantly from control ( data not shown ). the results indicate that blocking the k + binding site has no effect and therefore , that stimulation of na + , k + pump activity is not essential for the expression of the signature - type . colchicine is as potent as monensin at inducing the signature - type . under conditions employed in our experiments , colchicines caused the complete depolymerization of mts . thus , the effect is attributed to the fact that mt integrity was crucial for the normal processing of endosomal contents . when iar20 pc1 cells were treated with colchicine and then classified by factors , the equation solution advanced to 338 days in culture , over the 283 days obtained for control ( fig1 ). qualitatively , as well , the colchicine - induced phenotype resembled that induced by ionophores . the prevalence of filopodia (# 4 ) decreased , as had been observed in ionophore - treated cells . in one experiment , the factor # 7 value increased for the colchicines - treated sample indicating that knob - like structures at the cell edge were affected , as had been found in gramicidin - treated cells ( fig6 - table 2 ). the arrangement of mts was unaffected by ionophore exposure ( data not shown ), and so the general similarities in phenotype did not imply that a common mechanism mediated the cell &# 39 ; s response to the two classes of agents . two major differences were seen in the response , one in values of # 12 , a factor that reflects the slope at which the cell surface rises from the substratum . both factor # 12 and , in 1000 w cells , # 1 , a factor for massive extensions or invaginations in the two uppermost contours , increased in cells exposed to colchicine but not in those exposed to ionophores . the effect of mt - stabilizing drugs , such as paclitaxel , was also studied . paclitaxel caused bundling of mts , so that they were gradually excluded from the rest of the cytoplasm . in some cells types , it inhibited mt - dependent traffic of endosomes to the lysosome ( 13 ), whereas in others , internalized receptors were degraded at the normal rate in the presence of the taxane ( 14 ). paclitaxel impairs the movement of lysosomes along mts although to a lesser extent than mt - depolymerizing agents ( 15 ). the data suggest that the effects on endosome processing were cell - type specific and that paclitaxel might stimulate features of the signature - type in some cells . indeed , preliminary results show that the agent has this effect on iar20 pc1 cells , if it was administered at a high concentration over a 4 - h period ( heckman c a , j cell biol 111 : abs . 52 , 1990 ). when this phenotype is broken down in terms of factors , the cells exhibited changes in # 1 , # 7 , # 8 , and # 13 ( data not shown ). differences seen in factors # 1 , # 7 , and # 13 were not unique to paclitaxel , since similar effects were obtained with at least one sample exposed to another taxane . exposing the cells to a lower dose of paclitaxel for 2 h led to changes in factors # 1 , # 7 , and # 12 , and the values of the former two came to resemble those of transformed cells ( fig6 - table 2 ). nevertheless , the treatment failed to promote the signature - type of these cells , as the solution for mean value of predicted time was 304 days , compared to 283 for control ( fig1 ), a difference that was not statistically significant . similarly , no difference was found when the data were classified on the basis of single variables . weak based induce the signature - type without significant changes in any factor value . cells were exposed to the antimalarial drugs , chloroquine and quinidine sulfate , and solved for values of predicted time in culture . values of 315 and 332 days , respectively were found . these differed significantly from the control value of 283 days ( fig1 ). although quinidine had not been tested previously , a similar result had been obtained for chloroquine when classification was based on single variables ( 1 ). since weak bases freely enter and neutralize acidic compartments , the results confirmed the importance of proton concentration and ph in causing cells to adopt the signature - type . when samples treated with the weak bases were analyzed for significant changes in factor values , it was found that these compounds did not significantly change any of the factors used in classification ( fig6 - table ii ). phenotype reversal upon exposure to a combination of mt inhibitors ( ic ). certain taxanes , when administered simultaneously with an mt inhibitor , stabilized the mts against depolymerization . it was reasoned that , if the colchicines - induced shift to the signature - type required mt depolymerization , then treatment with an ic containing such a taxane would counteract the response to coichicine and maintain the normal type . to explore this possibility , iar 20 pc1 cells were to a combination of paclitaxel and colchicine . the results were unexpected , however , as the ic treatment reversed the signature - type pattern . the mean predicted time for the treatment sample was 265 days , versus 291 days for the control ( fig2 ) to determine whether this effect could be achieved by delivering colchicine together with a paclitaxel analogue , two such analogues and their ics were studied . however , the ic used initially was uniquely effective compared to those made up with other taxanes . exposure to baccatin iii , cephalomannine , or 7 - deoxytaxol alone failed to shift the samples &# 39 ; means to values that differed significantly from control . cells exposed to an ic made up with either of the latter two taxanes also failed to change their phenotype ( fig2 ). when values of individual factors were analyzed , both the taxane - and ic - treated samples were distinguished from the control by a difference in factor # 1 value . although there were few significant changes in cells treated with cephalomannine and colchicine , the ic containing 7 - deoxytaxol caused alterations in values of several factors ( data not shown ). in cells exposed to the former ic , # 13 underwent a shift to the value observed in transformed cells ( fig6 - table 2 ). this shift , which would have been expected to enhance the signature - type , did not lead to the sample differing significantly from control ( fig2 ). at the other extreme , cells treated with an ic of paclitaxel and colchicines could reverse their phenotype without exhibiting a significant change in any factor &# 39 ; s value ( fig6 - table 2 ). this suggested that small shifts in a number of factors must have occurred to account for the overall shift in classification . to investigate the reversal of the signature - type further , it was determined whether reversal could be induced by exposing 1000 w tracheal cells to paclitaxel and colchicine . the predicted time values of the samples were shifted only slightly compared to control . the signature - type of cells treated with this ic was 611 days compared to 638 days for control , a difference that was not statistically significant . when individual factors of the subset used for classification were analyzed , to determine whether their values were disproportionately affected , no significant differences were found . even when paclitaxel was used alone , it failed to elicit any change in the signature - type of 1000 w cells . this was true regardless of whether cells were exposed for the standard 2 - h interval or the exposure was lengthened to 5 h . thus , the data suggest that reversal of the phenotype by this ic was specific to iar20 pc1 cells . in further analyses , key variables contributing to # 4 , the factor accounting for the major fraction of transformation - dependent variability in the data , were investigated . it was reasoned that , if factor # 4 underwent a marginal change in value , underlying changes in one or more of the eight contour shape variables that were highly correlated with it would be found . indeed , six of their values differed significantly in paclitaxel - treated 1000 w cells , indicating that the boundary of the cell became more complex . four of the six were still changed in ic - treated cells , suggesting that these samples differed very little from those treated with paclitaxel alone ( fig7 - table 3 ). use of method to find therapies and examples of diseases useful with method of invention . the effects of taxanes on 1000 w cells were dissimilar to those found when the iar20 pc1 liver cell line was used ( deposited with the atcc under patent deposit designation no . pta - 4019 on jan . 30 , 2002 and found to be viable ). this cell line was taken from a deposit maintained by bowling green state university , department of biological sciences , bowling green , ohio , since prior to the filing date of the provisional application 60 / 280 , 053 . paclitaxel alone was capable of reversing the direction of change of six out of the eight above variables , compared to the direction they were shifted in transformation . these shifts in phenotype suggested that paclitaxel itself could reverse evidences of cancerous transformation . since the ic of paclitaxel and colchicine had similar effects to those of paclitaxel alone , the data show that this taxane , alone or in combination with colchicine , had the power to reverse certain features of the cancer cells . this was not the case for iar20 pc1 cells ( fig7 ). in these cells , the direction of change of one of the variables , csqd , was reversed in the ic - treated cells , moving in the opposite direction to that taken after treatment with the taxane alone . taxane treatment tended to smooth out the sharp features at the cell edge , whereas the ic enhanced the length and sharpness of such features . ics made up with taxanes other than paclitaxel , however , had a tendency to simplify the boundaries and therefore , these ics were totally unlike the ic containing paclitaxel . the importance of the differences described above can hardly be overestimated , because there is a novel principle reflected by the activity of an ic such as paclitaxel and vinorelbine , another mt - depolymerizing agent . in the case of many standard chemotherapeutic drugs , they are thought to merely act as poison to the cells . the mt - inhibitory drugs are a little bit more specific than most , as they target specifically the dividing cells of the patient &# 39 ; s body , working by poisoning the mitotic apparatus , which is used by the cell to complete distribution of its chromosomes to two daughter cells . the ics composed of mt - inhibitors , however , are working on a more sophisticated biological level . these combination agents work by inducing the process of apoptosis ( 84 ). apoptosis is a natural pathway of cell death , that is used normally in tissue reconstruction in order to clear away cells and tissues that are extraneous to further development of a fetus , new - born , or young animal . since the principle of triggering apoptosis by administering the ic to the colon cancer cells is wholly unknown , it is important to have an assay whereby a biological effect of the ic may be predicted . the present invention provides an assay which predicts the biological efficacy of an mt - inhibitor combination through a means that does not rely upon mitotic arrest . although the two cell lines used in the above - mentioned examples are from dissimilar organs and tissues of the body , they originate from the same tissue in embryonic development . along with the respiratory tract and liver , represented by the 1000 w and iar20 pc1 lines respectively , other organs derived from the endodermal layer include gall bladder , pancreas , colon , small intestine , urinary bladder , urethra , and thyroid gland . cancer cells from the epithelium of all these organs may be reversed in some of their properties when treated with taxanes . the above data provide evidence , however , that different cells from the same embryonic lineage respond very differently to paclitaxel ( see fig7 ). thus , it is possible that taxanes as sole agents and / or combinations of different mt - depolymerizing agents with taxanes may be effective chemotherapeutic agents for some of these organ and tissue types . evidence from medical studies on breast cancer patients suggests that an ic made up from vinblastine ( vinorelbine ®) and a paclitaxel analogue , docetaxel ( taxotere ®)), is an effective therapy for women who develop metastatic disease . prospective , randomized trials have now been done with this ic , and it has been found active ( 85 ). because of a necessity to test therapeutic treatments on patients with no known alternative that is more effective , the only data that exist to date are on patients with advanced disease . phase i data suggest that the ic used clinically , vinblastine and paclitaxel , is effective in patients with esophageal and lung cancer , as well as breast cancer ( 86 ). clinical tests of ic therapy as a primary treatment for breast cancer are now underway . the use of two mt inhibitors as a therapy is , therefore , in the process of being evaluated . since the breast tissue is derived from the embryonic layer known as ectoderm , the current developments suggest that use of taxanes in an ic are effective for tissues and cells arising from both endodermal and the ectodermal lineages . there is no experimental work which can be used as a basis on which to judge whether tissue from the third embryonic lineage , called mesoderm , are also susceptible to ic - induced reversal . it should be noted that these neoplasias were relatively differentiated in comparison to the iar20 pc1 cells , which formed anaplastic tumors when injected into host animals . the question whether the reversal of the cells &# 39 ; response to paclitaxel , when it was administered in the presence of colchicine , was an important indicator of the therapeutic effect of an ic was explored . the research was conducted by clinical practitioners working on human subjects with advanced cancer . differences in mt organization . in order to compare the cytoskeletal organization of cells treated with taxanes alone and those treated with an ic , exposed cells to the mt inhibitor ( s ) and then localized tubulin . baccatin iii had little noticeable effect , but the other taxanes elicited dramatic changes in mt organization . cephalomannine caused mts to aggregate in the form of arc - shaped bundles that partially encircled the nucleus ( fig3 ). paclitaxel - treated cells also contained large bundles of mts , as noted by others ( 16 , 17 ). in both cases , changes caused by the ic were more moderate . cells exposed to an ic containing cephalomannine appeared flatter , with more diffusely distributed mts , than those treated with cephalomannine alone . both samples , however , exhibited curving mts throughout the cytoplasm and at their edges . when cells were treated with an ic containing paclitaxel , bundles were less prominent and the exaggerated mts looked straighter and more delicate , but the bundles of mts themselves were organized similarly to those in paclitaxel - treated cells . in nearly every cell , an area could be detected where mts appeared to radiate away from a point in the cytoplasm . often , more than one of these areas was present so that mts formed a criss - crossed array in the cell periphery . originating from such points , the areas often radiated all the way out to the cell edge ( fig3 ). since cells treated with paclitaxel alone frequently had a fringe of straight mts extending out toward the cell edge but lacked criss - crossed arrays , these appeared unique to the ic - treated cells . varying the concentration of the paclitaxel in the ic between 6 and 18 μm did not change the mt organization compared to the example shown in fig3 . the data show that colchicine had a striking effect on the arrangement of mts when it was in an ic with paclitaxel , but failed to substantially alter their arrangement when it was delivered together with cephalomannine . to determine whether there was any difference in the sites where mt bundles formed after the two types of ic - treatment , the location of the bundles were tabulated . they were found on both sides of the nucleus in 71 % and 56 % of cells treated with cephalomannine and paclitaxel alone , respectively . the comparable values for ic - treated cells were 59 % and 62 %, suggesting that there was a tendency for mts to aggregate around more than one mt - organizing center in the presence of either taxane . control cells had a concentration of mts on one side of the nucleus , where they appeared to converge at the mt - organizing center . over 70 % of the control cells exhibited this arrangement , while the remainder showed two sites where the mts were convergent . this is consistent with the belief that net recruitment of tubulin into mts and bundling of the mts occurred , regardless of which ic was administered . in cells treated with paclitaxel and colchicine , about one - third fewer mts appeared to radiate from the mt - organizing center than in either paclitaxel - treated or control cells . therefore , it is believed that the suppression of mt - organizing centers around the nucleus is a distinct feature of ic - treated cells . relationship of growth factor signaling to the signature - type . in other cultured model systems , insulin stimulated the rate of fluid - phase endocytosis ( 18 ). therefore , the ambient concentration of insulin was varied to see whether this would perturb endocytic processing and hence , change the phenotype of 1000 w cells . the growth medium used for these cells contained supra - physiological levels of insulin , as it was routinely supplemented with 0 . 1 μg / ml insulin ( approximately 10 nm ). in the range about 1 nm , insulin affects cells through its ability to bind to insulin - like growth factor receptors . to determine whether shape features were perturbed by elevated signaling , cultures were grown separately in media containing three different levels of insulin . when the values of mean predicted time were solved , there were no significant differences among samples grown at levels between 0 . 1 and 10 μg / ml ( fig4 ). when cells cultured in replicate dishes were given a short - term exposure to colchicine , the signature - type was still created regardless of supplementation level . however , samples supplemented with the lowest level of insulin showed the most significant difference in the duncan multiple range test . in a second experiment , the average solution of a colchicine - treated sample was 689 days , versus 636 for the control ( data not shown ). two samples derived from a culture with 1 μg / ml level of supplementation still showed values of 687 and 638 days in culture respectively ( fig4 ). these samples also differed at p ≦ 0 . 05 , as did cells supplemented with 10 μg / ml levels . the probability of finding a value this large , based on random error alone , was p ≦ 0 . 001 . the value of the difference between to treated and controls was evaluated by the z alpha / 2 value , which was 4 . 23 in the first pair of samples . the corresponding values for cells cultured in medium and high insulin concentrations were z alpha / 2 = 2 . 97 ( p ≦ 0 . 003 ) and 2 . 22 ( p ≦ 0 . 03 ). thus , the significance of the difference between treated and control groups declined as the insulin concentration increased . the mechanism of action of the tumor promoter depends on its ability to substitute for an endogenous second messenger , diacylglycerol , and thereby activate certain members of an enzyme family known as protein kinase c . the quantitative shape phenotype of cells treated with pma resembled the phenotype of bona fide cancer cells . the effect of pma on this phenotype was transient as explained above . when the shape phenotype was dissected into components by relating different variable &# 39 ; s values to shape features , several of the altered values appeared to rely upon a declining number of sharp features , such as filopodia and microspikes , at the cell edge . filopodia and microspikes are in turn regulated by a gtpase ( 79 , 80 ) of the rho family , cdc42 , which modulates actin architecture . although data suggest that pma counteracts the rearrangement of actin into filopodia and microspikes , there is no known link of cdc42 to any pma - responsive isozyme of protein kinase c . further , over expression of an active mutant of cdc42 itself did not restore the phenotype to that of a normal cell ( data not shown ). however , cdc42 &# 39 ; s contribution to biological effects , such as neuronal growth cone development , is antagonized by activating another gtpase , rho itself ( 81 ). a third gtpase of the rho family , rac , which regulates ruffling , is likewise antagonized by rho activation . additionally , p21 - activated kinase ( pak ) mediates the effects of cdc42 and rac on actin architecture and promotes formation of filopodia and focal contacts , while antagonizing rho function ( 82 ). this led to further investigation whether overexpression of pak or inhibition of rho activity would enhance the formation of sharp features . 1000 w cells from respiratory tract epithelium were cultured in slide chambers containing fiduciary marks and the pak gene was injected along with a gene for green fluorescent protein ( gfp ). the shape phenotype was analyzed in cells that were first prepared for scanning electron microscopy and then relocated in the field of view at low magnification . to explore where elimination of rhoa activity might enhance the process of filopodia or microspike formation , a dominant negative mutant rhoa ( dnrho ) along with gfp was introduced . this genetic construct could inhibit rho - mediated effects by competing for the guanine nucleotide exchange factor ( s ) that activates endogenous rho . therefore , the results obtained by overexpressing dnrho were expected to resemble those obtained when overexpressing the gtpases , cdc42 or rac . cells overexpressing gfp and / or another gene construct were identified by epifluorescence microscopy . points representing the perimeter were extracted from a tif file and analyzed mathematically ( 7 ). data sets made up of at least 30 cells were subjected to statistical analysis . four types of contours extracted from such cells and tif files are shown in fig8 . the variables that are heavily weighted in the computation of factor # 4 include shpf , ptom , csqd , frnc , wdth , maxp , pshr , and ashr . the values of these descriptors increased or decreased in transformed and pma - treated cells . other descriptors that were less heavily weighted included mdal , nonc , alti , fine , cavs , acav , and lcav . some of these values were changed on cells that were co - injected with dominant negative rho and pak ( fig9 ). [ 0164 ] fig9 shows the results of tukey &# 39 ; s test on values of csqd and nonc for cells overexpressing genes for the proteins designated in fig8 . in addition , the pak enzymatic blocker ( pkb ) and rho gtpase activity blocker ( cbc3 ) were introduced into certain cells . samples enclosed by the same bracket are statistically indistinguishable . the resolution of cell samples overexpressing gfp , pak and dominant negative rho ( pkr ) indicate that sharp features have been reintroduced into cells such as those expressing gfp alone . in addition to the method summarized in fig9 being useful for basic research on the aberrations cancer cells , the method is also useful for assaying potential chemotherapy and chemopreventive drugs . key values of several shape factors ( f ) are used in an equation to evaluate the cell &# 39 ; s phenotype , for example : yhat = 242 . 01 + 26 . 5 * f 1 − 11 . 7 * f 3 − 49 . 9 * f 4 + 21 . 2 * f 5 + 19 . 7 * f 7 + 14 . 5 * f 8 + 29 . 8 * f 12 − 27 . 7 * f 13 since both of the cell lines used as subjects in the assay respond to taxane singly or in a combination with colchicine ( fig7 ) by acquiring sharper and / or longer features at the cell edge , variations in drugs composed from taxanes and mixtures of drugs including taxanes to achieve this effect are also within the scope of the present invention . reversal of the whole cancer cell phenotype is observed more rarely than the reversal of a single feature such as sharp features ( f4 ). since , in one cell line that is a subject of the assay , the overall reversal effect has been observed to be caused by exposure to one ic , substitution of another mt - depolymerizing agent for colchicine is also within the contemplated scope of the present invention . further , synthesis of a portmanteau drug by combining paclitaxel and colchicine , and subsequently testing it as a chemotherapy or chemopreventive drug in humans is also within the scope of the present invention . still further , synthesis of a portmanteau drug , combining paclitaxel and any other mt - depolymerizing agent , in the hope of producing a chemotherapeutic or chemopreventive drug for cancer is also within the scope of the present invention . the index of efficacy for any such drug or drug combination is the results of an assay for phenotype based on cells &# 39 ; mass distribution and geometrical shape features . another aspect of the present invention is , therefore , a practical method for detecting the efficacy of any drug or drug combination against cancer . a flowchart showing the preferred method of image processing and segmentation is provided ( fig1 ). the extraction of latent factors from shape data is done in such a way that the new variables are qualitatively related to features which can be perceived by viewing the cells . the use of a large database of values for shape variables , as done herein , is desirable if factors are to be generated which approach this ideal . the definitions and names of highly correlated shape variables that contributed to a data set of exemplary factors are provided below . here , the first 13 out of 20 factors could be given intuitive descriptions based on the variables they incorporated . although certain factors beyond the 13th factor could also be given descriptive names , they became increasingly more contour - specific and more specific to a single kind of shape feature . such intuitive factors can be used in interpreting alterations in cell shape characteristics that may be caused by exposing cells to a test drug or biological agent . factor # 1 ( having coarsely protruding or dissected areas in the higher contours ) factor # 2 ( having more or bigger protrusions in the second and third contours ) factor # 5 ( having mass displacement ( bulky projections or enlarged invaginations ) at cell edge ) contour # 1 arat , sdwd , mdal , sdcd , sdfd , fine , ashr , acav , lcav factor # 7 ( showing any regular scalloped structure at the cell edge ) factor # 12 ( showing a steep rise of the cell edge off the substrate ( rounding - up )) in certain preferred embodiment , the values of selected factors will be solved and used to predict properties or characteristics of any sample of cells presented as an unknown . throughout this application various publications are referenced by numerals within parenthesis . full citations for these publications may be found at the end of this application , preceding the claims . the disclosure of these publications , and all publications mentioned herein , in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains . while this invention has been described with emphasis upon preferred embodiments , it would be obvious to those of ordinary skill in the art that preferred embodiments may be varied . it is intended that the invention may be practiced otherwise than as specifically described herein . accordingly , this invention includes all modifications and claims spirit and scope of the appended claims . 1 . heckman , c . a ., plummer , h . k ., iii and runyeon , c . s ., persistent effects of phorbol 12 - 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