Connexin 37 profoundly slows cell cycle progression in rat insulinoma cells

Abstract

In addition to providing a pathway for intercellular communication, the gap junction-forming proteins, connexins, can serve a growth-suppressive function that is both connexin and cell-type specific. To assess its potential growth-suppressive function, we stably introduced connexin 37 (Cx37) into connexin-deficient, tumorigenic rat insulinoma (Rin) cells under the control of an inducible promoter. Proliferation of these iRin37 cells, when induced to express Cx37, was profoundly slowed: cell cycle time increased from 2 to 9 days. Proliferation and cell cycle time of Rin cells expressing Cx40 or Cx43 did not differ from Cx-deficient Rin cells. Cx37 suppressed Rin cell proliferation irrespective of cell density at the time of induced expression and without causing apoptosis. All phases of the cell cycle were prolonged by Cx37 expression, and progression through the G(1)/S checkpoint was delayed, resulting in accumulation of cells at this point. Serum deprivation augmented the effect of Cx37 to accumulate cells in late G(1). Cx43 expression also affected cell cycle progression of Rin cells, but its effects were opposite to Cx37, with decreases in G(1) and increases in S-phase cells. These effects of Cx43 were also augmented by serum deprivation. Cx-deficient Rin cells were unaffected by serum deprivation. Our results indicate that Cx37 expression suppresses cell proliferation by significantly increasing cell cycle time by extending all phases of the cell cycle and accumulating cells at the G(1)/S checkpoint.

Fig. 1.

Connexin 37 (Cx37) is significantly induced, forms occasional plaques, and appears in the Triton X-100-insoluble fraction of cell homogenates. A–C: cells were stained with Hoechst dye (nuclear stain, blue) and immunostained with Cx37-18264 antibody (Cy3-conjugated secondary, pink); overlays of the two images are presented. Cx37 labeling is evident in nearly all cells induced with either 0.25 μg/ml (B) or 2 μg/ml (C) doxycycline, but it is not detected in noninduced (A) cells. Obvious gap junction plaques were infrequent at both doxycyline concentrations; nevertheless, punctae at regions of cell-cell contact were occasionally observed (inset in C; magnification is 3.5-fold higher than in A, where scale bar represents 20 μm). D: Western blot of total [whole cell (WC)] and Triton-insoluble (TX) protein isolated from induced (Dox+) and noninduced (Dox−) cells. Doxycycline induced a significant increase in Cx37 expression (compare WC Dox+ and WC Dox− lanes). Cx37 was also readily detected in the Triton X-100-insoluble fraction. (WC−, WC+, and TX+ lanes were loaded with 33, 21, and 33 μg protein, respectively.)

Fig. 2.

Functional gap junctions and channels are formed by rat insulinoma (Rin) cells induced to express Cx37 (iRin37). A: mean conductance and individual data points for junctions formed by pairs of iRin37-F and -H cells. Means were not different despite different expression levels (see supplemental data Fig. 1S). Data for iRin37-F clone were derived from 7 different cultures on 7 distinct days, and data for iRin37-H were from 2 different cultures and days. Multiple coverslips of cells were used each day. B: single-channel record and all points histogram from a pair of iRin 37-F cells. Conductance of the fully open channel was ∼350 pS, and at least two substates (100 and 180 pS) were observed.

Fig. 3.

Proliferation of Rin cells was suppressed by Cx37 but not by Cx40 or Cx43 expression. In the absence of Cx37 expression, iRin37-F and -H cells proliferated comparably to the parental iRin cells (A). However, when Cx37 expression was induced (B), proliferation of the 37-F and -H clones was suppressed. Constitutive expression of rat Cx40 (Rin40) or rat Cx43 (Rin43) did not affect cell proliferation, despite the absence (C) or presence (D) of doxycycline. Each point represents the average of 3 wells of cells; where error bars (SE) are not evident they are obscured by the symbols.

Fig. 4.

Cx37-induced suppression of proliferation is density independent and has a rapid onset. iRin37-F cells were plated at 3 × 10⁴ cells per well. Doxycycline was added to one third of wells 24 h later (Dox+ data) and to another third 12 days later (Dox: 12d−, 9d+ data); the remaining wells were untreated (no Dox). Cell number was monitored at 3-day intervals for 21 days for all treatment groups. Proliferation of cells exposed to doxycycline was suppressed irrespective of cell density at the time of induction.

Fig. 5.

Cx37, Cx40, and Cx43 are comparably localized and obvious gap junction plaques are largely absent in iRin37-F, Rin40, and Rin43 cells. Shown are iRin37-F (A–C), Rin40 (D–F), and Rin43 (G–I) cells. Cx-specific labeling is shown in A, D, and G and as overlays (pink) with Hoechst nuclear stain (blue) in B, E, and H; phase contrast images of immunostained fields are shown in C, F, and I. Scale bar represents 20 μm and applies to all panels.

Fig. 6.

Cell cycle position of iRin37-F cells induced or not to express Cx37 for 48 h. Although a sub-G₁ peak, indicative of apoptosis, was sometimes observed in all types of Rin cells (inset shows iRin cells), such a peak was not predictably observed in iRin37-F cells (induced or not). In these samples, the percentage of cells in G₀/G₁, S, and G₂ for the noninduced iRin37-F cells was 71.8%, 17.4%, and 10.8%, respectively, and for the induced iRin37 cells was 76.18%, 13.65%, and 10.17%, respectively.

Fig. 7.

Cx37-expressing cells do not exit the cell cycle into G₀. iRin37-F cells were plated on glass coverslips, induced (C and D) or not (A and B) to express Cx37 for 48 h, and stained to reveal PCNA expression (brown), which is absent in G₀ cells. Cells were counterstained with hematoxylin (blue in B and D) and observed at ×10 and ×40 (insets). PCNA expression was clearly evident in noninduced cells (A) and induced cells (C). No nonspecific staining was observed in cells exposed only to secondary antibody (B and D). Data are representative of 4 experiments. Scale bar (in D) corresponds to 50 μm for ×10 images and to 9 μm for insets.

Fig. 8.

Doubling time is significantly increased in Cx37-expressing cells. A and B: pooled data from iRin and noninduced iRin37-F cells (A; iRin37−) and induced iRin37-F cells (B; iRin37+). Proliferation of Cx37-expressing cells is suppressed by nearly 23-fold; note the difference in y-axis scaling between A and B. iRin and iRin37+ data derive from 16 experiments, iRin37− from 14; time points with reduced sample sizes are noted in the figure. Logarithmic plots (insets) reveal exponential growth between days 3 and 15 for iRin and iRin37− cells; the iRin37+ cells transition between two periods (6–15 and 18–21 days) of exponential growth. C: calculated doubling time for each cell type for their logarithmic growth periods; a 6-day window of analysis was used, in which the 6th day is noted on the abscissa.

Fig. 9.

Cx37 expression continues in virtually all iRin37-F cells throughout a 21-day proliferation assay. A and C: cells stained with Hoechst dye. B and D: Cx37 expression for the same cells. Images in A and B were taken 24 h after induction, and in C and D, 21 days after induction. Note the increase in cell number between days 1 and 21. Scale bar represents 9 μm.

Fig. 10.

Cell cycle progression and response to serum starvation differ in Cx37-expressing (iRin37-F) vs. Cx37-deficient (Rin) cells. Shown are the percentages of the cell population in G₁ (A), S (B), and G₂ (C) following 72 h in 10% serum (open symbols on the left in each graph) or 0% serum (0-h time points) and as a function time following restoration of 10% serum to the serum-deprived cells. All cells were treated with doxycycline for the 24 h preceding the 0-h time point and throughout the remainder of the experiment. *Significant difference from non-serum-deprived cells (G₁ and S, in iRin37 only). #Significant difference from the 0-h time point. †Significant difference between cell types at the indicated time point.