Most mitotic HeLa cells divided into two daughter cells with half the volume of the parent; no additional reduction in volume was detectable during late telophase or the early part of G1. Synchronous growth throughout the next generation cycle was exponential, without an additional and sudden rise in volume being detectable as cells entered M-phase. Since the wet weight/dry weight ratio and the protein per unit mass of cells remained constant throughout the cycle, measurements based on volume changes accurately reflected growth parameters and cell water content. Nuclear magnetic resonance (n.m.r.) t1 (spin-lattice) proton relaxation times for intracellular water for M-phase cells measured at both 32 MHz (25 degrees C) and 80 MHz (37 degrees C) in a Bruker spectrometer had the same values as normal interphase cells. Furthermore, cells arrested in metaphase by nitrous oxide or alkaloids gave the same t1 values. Artificial manipulation of cell hydration by adjustment of the tonicity of the external medium led to t1 values that correlated well with the level of intracellular water. On this basis, a 40% increase in hydration raised t1 values by a factor of 1.29 at 32 MHz and 1.5 at 80 MHz. This is considerably less than the increase by a factor of 1.9 in t1 time reported with the 40% rise in cell water accompanying mitosis, measured at 30 MHz and 25 degrees C under isotonic conditions by others. The protein-synthesizing ability of hydrated cells was reduced to only half the normal level after a doubling of intracellular water. The data from these several different analyses, taken together, strongly indicate that the water content of mitotic cells is very similar to that of interphase cells, and that certain features unique to the mitotic phase of the cell cycle cannot be ascribed to an elevated (free) water content.

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