Proliferation assays were carried out to ascertain whether the size of soft agar colonies ( Figure 5 and 6 ) was proportional to the rate of proliferation in vitro calculated from changes in cell number as a function of time. As shown in Figure 7 , 253T-NT cells expressing wild type CEACAM1-4S proliferated 2.2 times faster than 253T-NT cells carrying the empty vector and from 1.4-2.2 times faster than cells transfected with glycine mutants. Consistent with their rapid growth in soft agar, 253T-NT cells expressing CEACAM1-4S with single or double tyrosine mutants proliferated at rates that were 1.4-1.6-fold higher than cells with wild type CEACAM1-4S. Taken together, these data suggested that the CEACAM1-4S transmembrane domain was controlling interactions involved in growth under anchorage independent conditions, interactions that were altered by GXXXG or tyrosine mutations.