Biomedical subjects

M P Quinlan

Publications and source records attributed to M P Quinlan.

At least 19 recordsLinked to original sources

PubMed2001

Identification of genes involved in epithelial-mesenchymal transition and tumor progression.

The adenovirus E1A12S gene product (WT12S) immortalizes epithelial cells and they retain their differentiated characteristics, but certain mutants cannot do the latter. Characterization of mutant immortalized epithelial cells indicated that they had undergone epithelial mesenchymal transition (EMT). Coexpression of V12ras with WT12S leads to benign tumors, but to malignant tumors with 12S mutants. Since EMT is critical for tumor progression, identification of the molecular mechanisms involved should elucidate novel therapeutic targets. To this end, representational difference analysis (RDA) was used to identify cDNAs upregulated in the mutant cell line. Thirty-five differentially expressed mRNAs were identified and classified into several functional categories, including nine novel cDNAs. Among the 26 known cDNAs, extracellular matrix and related proteins made up the largest group of differentially expressed genes, followed by growth factors and receptors and transcription factors. There was also an ion transporter, a cytoskeletal protein, glycosylation and amidinotransferase enzymes and proteins with unknown functions. Some of the known genes have previously been associated with EMT and/or tumor progression and thus served to validate the system to obtain the desired target genes, while other cDNAs are newly linked with dedifferentiation/malignancy. Array analyses indicated that the cDNAs were specifically upregulated in invasive or metastatic tumors, especially of breast, uterus and lung, suggesting their involvement in the progression of these tumors.

Adenoviridae↗

PubMed2000

While E1A can facilitate epithelial cell transformation by several dominant oncogenes, the C-terminus seems only to regulate rac and cdc42 function, but in both epithelial and fibroblastic cells.

Epithelial and fibroblast cells were differentially susceptible to transformation by oncogenic src, ras, mos, raf, rac, and cdc42 and the influence of adenovirus E1A. In contrast to NIH 3T3 cells, which are easily transformed by all the oncogenes tested, epithelial cells were more resistant to transformation by the same oncogenes. Transformation efficiency of both primary and immortal epithelial cells by E1B, V12ras, v-src, v-raf, and v-mos was increased by cotransfection of E1A 12S, which enables these cells to overcome the M1/M2 mortality blocks, which are not present in NIH 3T3 cells. NIH 3T3 cell transformation by these oncogenes was not altered by E1A. Although V12cdc42 or V12rac1 alone could produce foci on NIH 3T3 cells, morphological conversion was observed only in the presence of a hypertransforming E1A mutant and not WT E1A. Epithelial cells were not transformed by V12cdc42 or V12rac1, even in the presence of WT or mutant E1A, but could be transformed by coexpression of mos/raf and rac/cdc42, and the resultant phenotype was affected by the E1A C-terminus. Hypertransformation, which has previously been reported with ras and E1A C-terminal mutants, turns out to be due to a synergy with rac/cdc42, but not ERK/MAPK or PI3K ras effectors. Like V12rac, expression of the E1A hypertransforming mutant resulted in the upregulation of vinculin and VASP, concomitant with the altered organization of the actin cytoskeleton in these cells. The results show that in addition to requiring abrogation of M1/M2 mortality blocks, primary epithelial cells require activation of the ERK MAPK cascade and rearrangement of the actin CSK to achieve transformation. In addition, the E1A C-terminus regulates rac/cdc42 function in both epithelial and fibroblast cells to affect the extent of transformation progression.

3T3 Cells↗

PubMed1999

Rac regulates the stability of the adherens junction and its components, thus affecting epithelial cell differentiation and transformation.

We have previously reported that activated rac (V12rac) can bring about hypertransformation of ras-transformed epithelial cells, which can be suppressed by the dominant negative form of rac (N17rac). Starting with primary epithelial cells, a series of cell lines expressing wild type (WT) or mutated forms of ras or rac were generated and analysed for their adhesive function and expression and association of adherens junction (AJ) proteins. Normal, primary epithelial cells were self-adhesive and expressed AJs that were very stable. The expression of constitutively active ras resulted in a decrease in, but not loss of, cell-cell adhesion, with concomitantly decreased stability of AJ components. This was extremely exacerbated by the co-expression of constitutively activate rac, but was suppressed by dominant negative rac, which resulted in increased cell-cell adhesion and extremely stable AJs. alpha-catenin also failed to associate with E-cadherin-beta-catenin complexes in cells expressing V12rac. Expression of V12rac resulted in the loss of epithelial morphology. The extent of transformation of each cell type corresponded to the stability of the respective AJ complexes. Thus, rac seems to be involved in regulating the stability of AJs, which promote epithelial cell differentiation, and consequently, modulating tumor progression.

Animals↗

PubMed1999

Efficacy of supplementary intracameral lidocaine in routine phacoemulsification under topical anesthesia.

OBJECTIVE: To determine whether the routine use of supplementary intracameral lidocaine has any benefit over topical anesthesia alone when performing phacoemulsification surgery. DESIGN: A prospective single-center, randomized, double-masked, clinical trial. PARTICIPANTS: A total of 204 patients undergoing phacoemulsification surgery with lens implantation under planned topical anesthesia. METHODS: Patients were randomly allocated to receive either topical anesthesia plus 0.5 ml intracameral balanced salt solution or topical anesthesia plus 0.5 ml preservative-free 1% intracameral lidocaine. MAIN OUTCOME MEASURES: On the day after surgery, patients were asked to document the discomfort they had experienced using a visual analog scale. Intraoperative discomfort, postoperative discomfort, and discomfort caused by the microscope light were assessed. RESULTS: Multiple linear regression analysis did not show any significant relationship between the use of intracameral lidocaine and either intraoperative (P = 0.34) or postoperative (P = 0.45) pain scores. There was a small reduction in the discomfort caused by the operating microscope when intracameral lidocaine was used (P = 0.04). CONCLUSIONS: In this study, the routine use of intracameral lidocaine as a supplement to topical anesthesia was shown not to have a clinically useful role.

Aged↗

PubMed1999

Prolonged survival of adult rat pancreatic islets transfected with E1A-12S adenovirus.

It has been reported that various mutants of the E1A-adenovirus can activate quiescent differentiated cells to start proliferating. The aim of this study was to determine whether transfection with E1A-12S could extend the life span and functionality of pancreatic islets in culture. Rat pancreatic islets were isolated and transfected with retrovirus containing the adenovirus E1A-12S, E1A-13S, or control vectors. Transfection with the retroviral E1A-13S mutant produced extensive islet necrosis compared with nontransfected islets. Islets transfected with the control E1A mutant Ad5-dl312 vector (containing no E1A-12S or E1A-13S segments) were similar to nontransfected islets in their characteristics. We found that the E1A-12S transfected islets maintained greater viability, insulin granule structure, and glucose-induced insulin responsiveness over a 6-week period compared with mock or control islets. At 6 weeks of culture, the E1A-12S transfected islets also had fewer apoptotic cells compared with nontransfected islets. These data suggest that adenovirus E1A-12S can extend the functional life span of cultured rat pancreatic islets.

3T3 Cells↗

PubMed1999

Length of pregnancy in African Americans: validation of a new predictive rule.

This study evaluated whether a new predictive rule is more accurate for estimating the length of pregnancy in African Americans than Nägele's rule, the accepted standard. After identifying women in early pregnancy, telephone interviews were conducted to obtain information about 16 previously established determinants of gestational length. Based on these data, a linear multivariate regression model was used to predict an estimated delivery date (EDD) for each mother. In addition, the EDD was determined using Nägele's rule. Later, the actual delivery date was compared with the EDD predicted by the new rule and with the EDD predicted by Nägele's rule. Each pregnancy was assigned to its better prediction group, either the new rule's group or the Nägele's rule group. Fifty-seven pregnancies were identified prospectively and monitored. The new rule predicted the actual delivery date more accurately in 66% (37/56) of pregnancies, Nägele's rule was a better predictor in 34% (19/56) of pregnancies, and both rules were equally accurate in predicting the delivery date for one pregnancy. The new rule was more precise than Nägele's rule (P = .022) when the binomial distribution was used. When using the linear regression model rule, a more accurate EDD can be determined for African-American women. Moreover, it is possible to predict the risk of preterm delivery (those occurring > 3 weeks earlier than the EDD).

Adult↗

PubMed1999

Establishment of the circumferential actin filament network is a prerequisite for localization of the cadherin-catenin complex in epithelial cells.

With the adhesion molecules, the actin cytoskeleton controls cell-cell and cell-substrate interactions and participates in transmembrane signaling. The relationships between actin and adhesion complexes at the sites of adhesion have been well documented. Here we investigate by a series of studies whether a relationship exists between actin organization and the localization and function of the components of the cadherin-catenin complex (CCC) that participates in the cell-cell adherens junction. Reversible actin depolymerization reversibly affects the peripheral distribution of CCCs. Mutations in adenovirus E1A and the small GTPase rac1, but not Ha-ras, disrupt the circumferential, cortical actin filament (CAF) network and the targeting of CCC components to the cell surface. Disruption of actin stress fibers or microtubules does not interfere with CCC localization and function. Constitutive loss of the apical cortical actin ring results in epithelial cells in which components of the CCCs are found only in intracellular vesicles and never at the surface. A kinetic analysis of the de novo appearance of the CAF network and the CCCs at the cell surface was also conducted. When F-actin was dissolved, surface CCC components were internalized. Reestablishment of CAFs required about 4 h, during which time E-cadherin and alpha-catenin were found first in a juxtanuclear location and then in intracellular vesicles or post-Golgi carriers, similar to what was observed in cells expressing mutant E1A or rac1. Thus, disruption of preexisting CCCs resulted in their internalization and recycling to the Golgi. It was only after the regeneration of the filamentous actin ring beneath the cell surface that peripheral localization of CCCs was observed. A similar result was observed with dominant negative rac1. These data suggest that the status of cortical actin is assessed and transduced and thereby regulates the transport and delivery of cadherin and catenins to the cell surface.

Actin Cytoskeleton↗

PubMed1998

The C terminus of E1A regulates tumor progression and epithelial cell differentiation.

The E1A gene of adenovirus has been considered both a dominant oncogene and a tumor suppressor. It has been reported to induce epithelial cell but to prevent myoblast differentiation. E1A enables oncogenes that are unable to transform primary cells on their own to do so, yet suppresses tumor progression toward invasion and metastasis. To try to reconcile the seemingly, conflicting E1A phenotypes, we examined the expression of epithelial cell specific and characterizing proteins in immortalized or tumorigenically transformed primary epithelial cells expressing wild-type E1A or a C-terminal mutant that has lost tumor suppressive abilities. All the cell types continued to express cytokeratin. Epithelial cell morphology, social behavior, and growth characteristics were retained by cells expressing wild-type E1A, even in the presence of an activated ras oncogene. Mutant E1A-expressing cells were less well differentiated even in the absence of ras. They were specifically defective in cell-cell junctional complexes, such as tight and adherens junctions and desmosomes. There was also a preference for those actin structures prominent in fibroblasts: stress fibers and filopodia, while in the wild-type E1A expressing cells, cortical actin and circumferential actin filaments were dominant. Thus the E1A-mutant-expressing cells were already predisposed to a more advanced tumor stage even when they were only immortalized and not transformed. The results suggest the possibility that the C terminus of E1A may be involved in regulating epithelial mesenchymal transitions, which have previously been linked to tumor progression.

Actins↗

PubMed1998

Expression of the pRb-binding regions of E1A enables efficient transformation of primary epithelial cells by v-src.

Primary cultures of rat embryo fibroblasts have been shown to be resistant to transformation by dominant oncogenes such as v-src. We sought to determine if similar resistance is displayed by primary epithelial cells, and, if so, whether an immortalizing oncogene such as E1A could enhance transformation of primary epithelial cells by v-src. Transformation of primary rat epithelial cells by v-src was synergistically enhanced when E1A expression plasmids were cotransfected with a v-src expression plasmid. Foci were more numerous and observed earlier (9 to 14 days) with E1A plus v-src than with v-src alone (18 to 28 days). This cotransformation ability was abrogated by deletions in CR1 or CR2 of E1A, which encode the binding regions for the pRb family and are responsible for E1A-mediated cell cycle activation. Mutations in the p300 binding site or the second exon, which abolish immortalization, did not affect v-src cooperation, in contrast to ras and adenovirus E1B. While kinase activation was required for growth in soft agar, differential activation of Src kinase did not correlate with transformation efficiency. Cell morphology and actin structures were not dramatically impacted by E1A expression; thus, hypertransformation, as previously described for ras cotransformation, was not observed with v-src and second-exon mutants of E1A. However, growth rates for cells expressing both E1A and v-Src were higher than those for cells expressing only v-Src. These results suggest that functions involved in cell cycle activation encoded by E1A first exon can enhance v-src transformation of primary epithelial cells.

Actins↗

PubMed1998

Rac1 and extracellularly regulated kinase activation are sufficient for E1A-dependent cooperative transformation of primary epithelial cells, but progression can only be modulated by E1A or Rac1.

Ras transformation of fibroblast cell lines requires activation of multiple distinct signal pathways that act synergistically. E1A-ras cotransformation of primary epithelial cells is enhanced by distinct mutations in the second exon of E1A, resulting in "hypertransformation" and metastasis. The molecular and cellular differences in the in vitro properties of such transformed cells are characterized here. Hypertransformed cells grew faster and to higher saturation densities; had smaller, more refractile cell morphologies with pronounced actin microspikes; and were less adhesive when compared with wild-type (WT) E1A+ras-expressing cells. No significant differences were observed in extracellularly regulated kinase activity levels between the hypertransformed and WT transformed cells. Activated raf and Rac1 together were sufficient for transformation of primary epithelial cells with E1A, whereas neither alone was competent to cooperate with E1A. In the presence of activated ras and WT E1A, activated Rac1 expression effected all of the hypertransformation properties. Dominant-negative Rac1 expression was suppressive of the hypertransformation phenotype, including cell morphology, actin cytoskeletal structures, decreased growth rates, and increased adhesion. Thus, hypertransformation is not the result of extracellularly regulated kinase differences but can be effected by perturbations in Rac1 signals, as well as E1A 12S COOH-terminal mutants.

Actin Cytoskeleton↗

PubMed1998

Identification of a novel mechanism of regulation of the adherens junction by E1A, Rac1, and cortical actin filaments that contributes to tumor progression.

Transformation progression toward more malignant behavior often results from a loss of epithelial cell behavior, especially cell-cell adhesion. E1A cooperates with ras to transform primary epithelial cells such that they maintain epithelial cell differentiation, including the proper localization of adherens junctions (AJs). Second exon mutants of E1A 12S cooperate with ras to produce a more aggressively transformed phenotype, termed hypertransformation, that includes the loss of adhesion. Such hypertransformation can also be achieved by the addition of activated Rac1 to cells expressing wild-type E1A and ras, suggesting that actin reorganization may be important for the hypertransformed phenotype. Primary epithelial cells expressing hypertransforming mutants of E1A or V12Rac1 exhibit the loss of cortical actin filaments. In these cells, AJ complexes do not incorporate alpha-catenin, fail to associate with the cytoskeleton, and fail to localize to the plasma membrane, resulting in the destabilization of the AJ components and a loss of function. Loss of these epithelial cell characteristics predisposes these cells to a more malignant phenotype due to the loss of cell-cell adhesion. Taken together, these results suggest a novel mechanism of regulation of AJ function in tumor progression that involves the correct targeting of the AJ components, and this is affected by the status of cortical actin, which can be differentially affected by E1A or Rac1.

Actin Cytoskeleton↗

PubMed1997

Mutations in CR1 of E1A 12S yield dominant negative suppressors of immortalization and the lytic cycle.

The Adenovirus 5 E1A 12S gene is responsible for the establishment of immortalization of primary cells by Adenovirus. We have identified two mutants of 12S (30K and NTdl814), which, when coexpressed with wild-type 12S in primary baby rat kidney cells, were capable of suppressing the immortalizing function of the wild-type 12S gene, even when the mutant proteins were expressed at levels lower than wild type. 30K and NTdl814 did not affect the ability of the coexpressed 12S to activate the cell cycle, but have a suppressive effect on 12S-induced DNA synthesis and proliferation at late times in the immortalization pathway. Both the dominant negative mutants have a deletion in conserved region (CR)1 in the first exon of E1A, which encompasses one of the pRb-family binding regions. However, the mutants did not effect the binding of cellular proteins to full-length 12S. A suppressive effect on wild-type 12S was not observed with mutants that have lost any other region or function. In addition, expression of 30K, which is equivalent to the protein encoded by the 10S mRNA of E1A, inhibited E1A function in lytic cycle. Thus, loss of the CR1 seems to be a prerequisite for a mutant to have a dominant negative effect on E1A functions.

Adenovirus E1A Proteins↗

PubMed1997

Choroid plexus cysts: infant and early childhood developmental outcome.

OBJECTIVE: To determine the infant and early childhood developmental outcome associated with choroid plexus cysts diagnosed prenatally. METHODS: Between January 1990 and August 1995, 8270 women underwent second-trimester ultrasound examinations. All women whose fetuses were diagnosed as having choroid plexus cyst(s) underwent ultrasonographic detailed anatomy survey, were offered fetal karyotyping, and were followed with serial ultrasounds. Fetal karyotype, associated structural anomalies, maternal serum triple analyte screen, neonatal outcomes, and infant and early childhood developmental milestones were recorded. The children were followed subsequently, and developmental assessment was performed with a modified Denver II Developmental Screening Test. RESULTS: A diagnosis of choroid plexus cyst was made in 89 fetuses (1.1%). The mean (+/-standard deviation [SD]) gestational age at diagnosis was 18.2 +/- 1.9 weeks (range 15-22). The cysts varied in size and laterality, with a mean (+/-SD) size of 5.9 +/- 3.3 mm (range 1-23). Three of the 61 women who underwent testing for fetal karyotype (4.9%) had abnormal karyotypes identified. All three karyotypes were trisomy 18, and all three trisomy 18 fetuses had additional sonographic abnormalities. All 28 women who chose not to undergo fetal karyotypic analysis delivered phenotypically normal infants. Infant and childhood developmental follow-up was performed on 76 children with cysts diagnosed prenatally. The mean (+/-SD) length of childhood follow-up was 35.5 +/- 16.2 months (range 12-82). All 76 children were found to be developmentally normal by the Denver II Developmental Screening Test. CONCLUSION: These observations suggest that the finding of isolated choroid plexus cysts is not associated with delayed infant and early childhood development or an increased risk of abnormal karyotype. The presence of isolated choroid plexus cysts does not warrant intensive infant and early childhood follow-up.

Brain Diseases↗

PubMed1997

Immortalization of primary epithelial cells by E1A 12S requires late, second exon-encoded functions in addition to complex formation with pRB and p300.

Immortalization of primary cells is an early and important event in multistep tumorigenesis and is itself a multistep process. Adenovirus E1A 12S encodes an oncoprotein that can rescue cells from senescence and overcome apoptosis, leading to their immortalization. Five regions of 12S, located in both exons, are required for immortalization. Two regions in the first exon are necessary to activate the cell cycle, increase the number of population doublings, and overcome the M1 stage of mortality. However, extension of life span requires overcoming crisis or M2, which can be accomplished by the expression of the second exon. Several cellular proteins associate with the peptide encoded by the first exon of 12S including pRB, p107, p130, and p300. The importance of pRB-E1A and p300-E1A complexes in transformation is well established; however, their roles in 12S-mediated immortalization remain undefined. Results obtained from the present study using a panel of second exon immortalization-defective mutants demonstrate that formation of pRB-E1A and p300-E1A complexes is insufficient for immortalization of primary cells. We further demonstrate that the expression levels of another tumor suppressor protein, p53, also do not correlate with the inability of the mutants to immortalize. Thus, mutations in the second exon of 12S do not affect the early steps in the immortalization pathway. The second exon mutants are defective in performing a late function in immortalization, involving the reactivation of the cell cycle, indicating that it is a crucial event in immortalization.

Adenovirus E1A Proteins↗

PubMed1996

Induction of a complex between rasGAP and a novel 110 kD protein is required for immortalization of primary epithelial cells by the E1A 12S oncoprotein of adenovirus.

Although established cell lines can be transformed with oncogenic ras, primary epithelial cells cannot, but require the coexpression of an immortalizing oncogene, such as the E1A region of adenovirus. We have previously shown that immortalization of primary epithelial cells by E1A 12S requires the expression of five regions encoded by both the first and second exons of the gene. However, only three of these regions, located in the first exon, are required for cotransformation of primary cells with an activated ras oncogene. Thus, the expression of oncogenic ras is able to abrogate the need for the E1A function(s) encoded by the second exon that are required for immortalization. This suggested the possibility that the functions encoded by the second exon of E1A may involve or interact with the normal ras signal transduction pathway. The results described herein demonstrate that immortalization-competent 12S gene products induce the expression of a novel 110 kD protein, p110, that forms a stable complex with rasGAP. Failure to induce the p110-rasGAP complex results in the concomitant loss of ability of 12S to immortalize primary epithelial cells. The appearance of this complex parallels the expression of the 12S protein and is sensitive to the levels of E1A 12S. p110 induction is independent of the ability of 12S to activate the cell cycle and of the presence of adenovirus E1B and is not observed in the presence of the large T antigen of SV40. Thus, it is not a general response to proliferation or tumorigenic transformation, but rather seems to be specific to the immortalization function(s) of E1A 12S.

Adenovirus E1A Proteins↗

PubMed1996

Structural limitations of the Ad5 E1A 12S nuclear localization signal.

The Ad5 E1A 12S gene encodes an oncoprotein with the ability to immortalize and cooperate with other viral or cellular oncoproteins to transform primary epithelial cells. The immortalizing function is dependent on the protein's efficient localization to the nucleus. A five amino acid nuclear localization signal (NLS), Lys-Arg-Pro-Arg-Pro, has been identified at the extreme COOH-terminus. This signal is necessary but not sufficient for efficient nuclear localization. A mutational analysis has been undertaken to further characterize the 12S NLS. The individual amino acids of the signal appear to have varying functional relevance. The lysine residue (a.a. 239) and the first arginine residue (a.a. 240) are the most critical. Changing the second arginine (a.a. 242) to threonine or either proline (a.a. 241 or 243) to alanine marginally diminishes signal function. Replacing the 12S NLS with the SV40 large T antigen (LT) NLS does not measurably affect the protein's nuclear localization. Sequences directly upstream of the NLS have a significant role in the proper localization of the 12S protein as illustrated by inefficiently localized mutants that have deletions of these sequences. Analyses of these mutants using a monoclonal antibody that recognizes the COOH-terminal four amino acids of the NLS have revealed that their signals are probably masked. To further investigate the importance of protein context in signal function, several NLS insertion mutants were constructed. Two regions in the first exon with predicted high surface probabilities and no known functions were chosen as sites for NLS insertions. Neither a wild-type 12S- nor a SV40 LT-NLS was functional in any of the new locations, indicating that for 12S, positioning of the NLS in the protein is critical.

Adenoviridae↗

PubMed1996

E1A second exon requirements for induction of target cell susceptibility to lysis by natural killer cells: implications for the mechanism of action.

The E1A oncogene of adenovirus type 5 induces susceptibility of mammalian cells to lysis by natural killer lymphocytes (NK cells). It is unknown whether sensitization to NK killing is mediated directly by targeting effects of interactions between E1A peptides and cell surface MHC molecules or indirectly by an E1A activity that requires structural integrity of the oncoprotein. To discriminate between these hypotheses, rat and hamster cells expressing wild type E1A were contrasted with those expressing truncated products resulting from E1A termination or deletion mutations. Transfected rat cells, expressing truncated proteins from the E1A first exon that encodes MHC-binding peptides, remained resistant to lysis by NK cells, whereas cells expressing full-length E1A protein were highly susceptible to lysis. Studies of infected hamster cells showed that addition of either of two, nonoverlapping, second exon regions reconstituted cytolytic susceptibility induction by E1A. The results do not support the E1A-peptide-MHC hypothesis, since no single E1A peptide coding region was sufficient to convey cytolytic susceptibility to expresser cells. The data indicate that coordinate functions of the E1A first exon and redundant accessory regions in the second exon are required for E1A-induced susceptibility to NK killing.

Adenovirus E1A Proteins↗

PubMed1995

Efficient nuclear localization and immortalizing ability, two functions dependent on the adenovirus type 5 (Ad5) E1A second exon, are necessary for cotransformation with Ad5 E1B but not with T24ras.

Expression of adenovirus type 5 E1A 12S is sufficient to immortalize primary baby rat kidney cells, but another viral or cellular oncogene, such as E1B or T24ras, is necessary for complete transformation. The regions of 12S sufficient for T24ras cotransformation have been well characterized and are located in the first exon. The second exon is dispensable for ras cotransformation, although it contains a region which appears to modulate the transforming phenotype. The same 12S first exon regions important in ras transformation are also necessary for E1B transformation. Analysis of an extensive series of second exon deletion and amino acid point mutations demonstrated that mutations affecting either the efficient nuclear localization and/or the immortalizing ability of the 12S protein also prevented cooperation with E1B. In general, the entire C-terminal half of 12S, including the nuclear localization signal, was necessary for efficient cotransformation with E1B. In addition to the differences between T24ras and E1B regarding 12S regions necessary for cotransformation, the characteristics of E1B-cotransformed foci differed from those of T24ras. The E1B foci took longer to appear and had a much slower growth rate. No hypertransformed foci were produced with E1B cotransfections, and established E1A-E1B lines exhibited minimal growth in soft agar compared with that of E1A-T24ras lines.

Adenovirus E1A Proteins↗